WO2001064849A1 - Blood-brain barrier reconstruction model prepared by cocultivation - Google Patents
Blood-brain barrier reconstruction model prepared by cocultivation Download PDFInfo
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- WO2001064849A1 WO2001064849A1 PCT/JP2001/001017 JP0101017W WO0164849A1 WO 2001064849 A1 WO2001064849 A1 WO 2001064849A1 JP 0101017 W JP0101017 W JP 0101017W WO 0164849 A1 WO0164849 A1 WO 0164849A1
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- blood
- cell line
- brain barrier
- immortalized
- brain
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/08—Coculture with; Conditioned medium produced by cells of the nervous system
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/28—Vascular endothelial cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2503/00—Use of cells in diagnostics
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2517/00—Cells related to new breeds of animals
- C12N2517/02—Cells from transgenic animals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
Definitions
- the present invention relates to a blood-brain barrier remodeling model, more specifically, a rat-derived immortalized brain capillary endothelial cell line, an immortalized astrocyte cell line, and an immortalized brain capillary pericyte cell line.
- a method for creating a blood-brain barrier remodeling model by culturing, an immortalized brain capillary endothelial cell line with enhanced expression of a blood-brain barrier marker gene obtained by co-culture, and such a blood-brain barrier The present invention relates to a method of screening a substance promoting blood-brain barrier formation using an immortalized brain capillary endothelial cell line with enhanced expression of a model or a marker gene of the blood-brain barrier.
- the blood-brain barrier is a barrier that restricts the transfer of substances from the blood into brain tissue, thereby protecting the brain from harmful substances.
- Lipid-soluble substances such as nicotine, caffeine, and heroin can easily pass through the blood-brain barrier, but generally non-lipid-soluble substances such as polar substances and strong electrolytes are difficult to pass, but are required for brain metabolism. It is known that water-soluble substances such as glucose permeate the blood-brain barrier and are transported to brain tissue by carriers.
- brain capillary endothelial cells In the brain capillaries, adjacent endothelial cells form tight junctions called tight junctions and do not leak out from the gaps between cells, so substances that enter and exit the brain are in principle It has to pass through brain capillary endothelial cells, as mentioned above, brain capillary endothelial cells are not only nutrients to the brain but also fine Drugs are transported into the brain by various transport systems expressed in the alveolar membrane. Or, although its substance is largely unknown, the blood-brain barrier has a special physiological function in which a transport system that excretes metabolites and foreign substances of neurotransmitters from the brain to the circulating blood system works.
- brain capillary endothelial cells are co-cultured with astrocyte cells, It is known to enhance the ability of brain capillary endothelial cells to express GLUT-1 (Hayashi Y. et al., GLIA, 19, 13-26, 1997).
- astrocyte site cells used for co-culture are primary cultured astrocyte sites, they need to be prepared every time, and they are usually separated and cultured from rats one day after birth.
- there is a problem that it is different from mature rat astrocyte cells Swanson RA et al., J Neurosci., 17, 932-940, 1997).
- the first oral site cell lines include human-derived glial tumor cells (glial cells) such as KG-1—C, U251, and GI-1 and RCR-1 and C6.
- glial cells such as KG-1—C, U251, and GI-1 and RCR-1 and C6.
- GFAP glial fibrillary acidic protein
- C6 cells do not express GLUT-1 and GLAST, which are native Na + -dependent L_glutamic acid transporters in astrocytes, and are proteins expressed in nerve cells.
- a certain EAACl is expressed (PalosT. P. et al., Mol. Brain Res., 37, 297-303, 1996).
- Blood-brain barrier Although, according to this report, rat fetal astrocyte cells and human umbilical cord, Blood-brain barrier reconstruction model using vein-derived endothelial cells And it is difficult to prepare.
- Japanese Patent Application Laid-Open No. 11-314982 by the present inventors has established a transgenic mouse into which the large T antigen gene of the temperature-sensitive mutant SV40tsA58 was introduced.
- By co-culturing the brain capillary endothelial cell line with an astrocyte cell line established from a transgenic rat into which the large T antigen gene of the temperature-sensitive mutant SV40tsA58 was introduced It is disclosed that the ability of capillary endothelial cells to express GLUT-1 is enhanced.
- the brain capillary endothelial cell line and the astrocyte cell line used for co-culture differ in the animal species from which they are derived, they must be used in a strict sense as an optimal blood-brain barrier reconstruction model in a strict sense.
- the blood-brain barrier plays an important role in drug transport and brain metabolism, and various reconstruction models at the in-vitro mouth have been proposed to date. It has been considered difficult to create an optimal blood-brain barrier reconstruction model.
- An object of the present invention is to provide a centrally acting drug based on the blood-brain barrier permeation mechanism (anti-dementia drug, therapeutic drug for brain tumor, therapeutic drug for virus, psychotropic agent) Drugs targeting various receptors expressed on endothelial cells (cerebral microcirculation improving drug, therapeutic agent for cerebral edema) or disorders of brain capillary endothelial cells (cerebrovascular dementia and Alzheimer's dementia)
- An object of the present invention is to provide a blood-brain barrier reconstruction model that is extremely useful for screening targeted drugs. Disclosure of the invention
- the present inventors have conducted intensive studies to solve the above-mentioned problems, and have developed a rat brain capillary endothelial cell line TR-BBB13 (FEPMBP-68783), which is an immortalized cell line established from animals of the same species. ), Rat trastocyte cell line TR-AST9332 (FEPMBP-62283), rat brain capillaries
- TR-BBB13 Rat trastocyte cell line TR-AST9332 (FEPMBP-62283)
- FEPMBP-62283 Rat trastocyte cell line TR-AST9332
- rat brain capillaries The present inventors have found that an optimal blood-brain barrier remodeling model can be prepared by co-culturing with the cell line TR-PCT1 (FERMBP-7024), thereby completing the present invention.
- the present invention provides a blood-brain barrier remodeling model characterized by co-culturing a rat-derived immortalized brain capillary endothelial cell line and a rat-derived immortalized astrocyte cell line.
- Preparation method (Claim 1), an immortalized brain capillary endothelial cell line derived from a rat, an immortalized astrocyte cell line derived from a rat, and an immortalized brain capillary pericyte cell derived from a rat
- a method for preparing a blood-brain barrier remodeling model characterized by co-culturing with a strain (Claim 2), and an immortalized brain capillary endothelial cell line derived from a rat is a temperature-sensitive mutant SV40tsA.
- a blood-brain barrier remodeling model which is an immortalized brain capillary endothelial cell line derived from a transgenic rat into which a large T antigen gene has been introduced. Item 3) and the temperature-sensitive mutant SV 40 ts A58
- the blood-brain barrier according to claim 3, wherein the immortalized brain capillary endothelial cell line derived from the transgenic rat into which the large T antigen gene has been introduced is TR-BBB13 (FEPMBP-6873).
- a method for preparing a reconstructed model (Claim 4), and a method in which a rat-derived immortalized astrocyte cell line is transgenic into which a large T antigen gene of a temperature-sensitive mutant SV40tsA58 was introduced.
- 3. The method for preparing a blood-brain barrier remodeling model according to claim 1 or 2, which is an immortalized astrocyte cell line derived from a rat (claim 5), and a temperature-sensitive mutant SV40.
- the immortalized astrocyte cell line derived from the transgenic rat into which the large T antigen gene of tsA58 has been introduced is TR-AST932 (FEPMBP-6283).
- the method for preparing a blood-brain barrier remodeling model according to claim 2 which is an immortalized brain capillary pericyte cell line derived from a transgenic rat into which a T antigen gene has been introduced.
- An immortalized brain capillary pericyte cell line derived from a transgenic rat into which the large T antigen gene of the temperature-sensitive mutant SV40tsA58 was introduced was TR-PCT1 (FERMBP-7204).
- the blood-brain barrier according to any one of claims 1 to 8, which is a system co-culture.
- the present invention relates to a method for producing a construction model (claim 9).
- the present invention also provides a screening method using the blood-brain barrier reconstruction model according to any one of claims 1 to 9, wherein the test substance is immortalized during or before or after co-culture.
- a method for screening a substance that promotes or suppresses the formation of the blood-brain barrier which is characterized by measuring and evaluating the degree of expression of a marker gene at the blood-brain barrier by contacting with a cell line (claim 10);
- the blood-brain barrier A screening method for a promoting or inhibitory substance (Claim 11) or a screening method using the blood-brain barrier reconstruction model according to any one of Claims 1 to 9, wherein the screening method is performed during or after co-culture.
- the present invention also provides a blood-brain barrier marker obtained by co-culturing a rat-derived immortalized brain capillary endothelial cell line and a rat-derived immortalized astrocyte cell line.
- Immortalized brain capillary endothelial cell lines with enhanced gene expression (Claim 19), rat-derived immortalized brain capillary endothelial cell lines, and rat-derived immortalized astrocyte cell lines
- An immortalized brain capillary endothelial cell line having enhanced expression of a blood brain barrier marker gene which is obtained by co-culturing a rat-derived immortalized brain capillary pericyte cell line.
- an immortalized cerebral capillary endothelial cell line derived from a rat was transformed into an immortalized cerebral capillary derived from a transgenic rat into which the large T antigen gene of the temperature-sensitive mutant SV40tsA58 was introduced.
- Endothelial cell line Features to claim 1 9 or 0 Expression of the marker gene of the blood brain barrier described enhanced immortalized brain capillary endothelial cell line (according Item 21) and the immortalized cerebral capillary endothelial cell line derived from the transgenic rat into which the large T antigen gene of the temperature-sensitive mutant SV40tsA58 was introduced were TR-BBB13 ( 21.
- the immortalized ostium site cell line is a transgenic rat derived from the transgenic rat into which the temperature-sensitive mutant SV40tsA58 radio-T antigen gene has been introduced.
- the immortalized brain capillary according to claim 23, wherein the oral site cell line is TR-AST932 (FEPMBP-6282), wherein the expression of the marker gene of the blood brain barrier is enhanced.
- TR-AST932 FEPMBP-6282
- a vascular endothelial cell line (Claim 24) or a rat-derived immortalized cerebral capillary pericyte cell line is a transgenic rat into which the large T antigen gene of the temperature-sensitive mutant SV40tsA58 was introduced. 20.
- the immortalized cerebral capillary endothelial cell line (Claim 26) or the co-culture, the immortalized cerebral capillary endothelial cell line and the immortalized ostium site cell line in non-contact or immortalized state Culturing an immortalized cerebral capillary endothelial cell line, an immortalized ostium site cell line and an immortalized cerebral capillary pericyte cell line in a non-contact state;
- the blood-brain barrier marker gene is one or more genes selected from the group consisting of alkaline phosphatase gene, alginate meal transpeptidase gene, and G1ut1 gene.
- Immortalized cerebral capillary endothelial cell line with enhanced gene expression (Claim 30) and G1ut1 gene expression was enhanced 100-fold or more compared to single culture without co-culture
- An immortalized brain endothelial cell line having enhanced expression of a blood-brain barrier marker gene according to any one of claims 28 to 30 (claim 31).
- the present invention also provides a test substance and an immortalized brain capillary endothelial cell line in which the expression of a marker gene for the blood brain barrier according to any one of claims 19 to 31 is contacted, whereby the blood brain barrier is A screening method for a substance promoting or inhibiting the formation of the blood-brain barrier, which comprises measuring and evaluating the degree of expression of the marker gene (Claim 32); and a test substance and a substance permeating the blood-brain barrier or blood.
- a screening method for a substance promoting or inhibiting the formation of the blood-brain barrier which comprises measuring and evaluating the degree of expression of the marker gene (Claim 32); and a test substance and a substance permeating the blood-brain barrier or blood.
- a method for screening a substance promoting or inhibiting blood-brain barrier permeation which comprises measuring and evaluating the degree of permeation into brain capillary endothelial cells (Claim 33); a test substance; 9) contacting the immortalized brain capillary endothelial cell line with enhanced expression of the blood-brain barrier marker gene according to any one of 9 to 31.
- the blood-brain barrier penetration-enhancing substance (Claim 37) obtained by the screening method of the blood-brain barrier permeation-promoting or inhibitory substance, or the blood-brain barrier penetration-enhancing or inhibiting substance screening method of Claim 33.
- FIG. 1 is a diagram schematically showing a non-contact type co-culture method in the present invention.
- FIG. 2 is a view showing the results of ALP activity of a blood-brain barrier reconstruction model by co-culture according to the present invention.
- FIG. 3 shows the results of ⁇ GTP activity of the blood-brain barrier remodeling model by co-culture of the present invention.
- FIG. 4 shows G in the blood-brain barrier reconstruction model by co-culture of the present invention.
- FIG. 9 is a view showing a result of LUT-1 expression.
- the method for preparing the blood-brain barrier remodeling model of the present invention includes co-culturing a rat-derived immortalized brain capillary endothelial cell line with a rat-derived immortalized astrocyte cell line, Co-cultured with an immortalized brain capillary endothelial cell line derived from a rat, an immortalized astrocyte cell line derived from a rat, and an immortalized brain capillary pericyte cell line derived from a rat.
- the immortalized brain capillary endothelial cell line having enhanced expression of the blood brain barrier marker gene of the present invention includes a rat-derived immortalized brain capillary endothelial cell line and a rat-derived immortalized astrocyte.
- the immortalized cerebral capillary endothelial cell line, the immortalized ostium site cell line, and the immortalized cerebral capillary pericyte cell line derived from the above-mentioned rat are not particularly limited, but are each normal.
- a cell line established as an immortalized cell while retaining the functions and properties inherent in brain capillary endothelial cells, astrosite cells, and brain capillary pericytes is preferable.
- the method of establishing these immortalized cell lines is not particularly limited.For example, immortalized cells obtained from transgenic rats into which the large T antigen gene of the temperature-sensitive mutant tsA58 of SV40 has been introduced.
- Brain capillary endothelial cell line, immortalized astrocyte cell line and immortalized brain capillary pericyte cell line can control their growth by temperature conditions, i.e. permanent at 33-37 ° C It is preferable because it retains the proliferative ability and stops the growth at 39 ° C., so that the expression of differentiation traits specific to cells can be controlled.
- the immortalized brain capillary endothelial cell line derived from the transgenic rat into which the large T antigen gene of the SV40 temperature-sensitive mutant tsA58 has been introduced includes the blood-brain barrier enzyme.
- Cell lines TR-BBB1 and TR-BBB that express the lipophilic enzyme phospholipase (ALP) ⁇ adal mirmir transpeptidase ( ⁇ GT GT) and the hexose transporter GLUT-1 5, TR—BBB 6, TR—BBB 11 and TR— ⁇ ⁇ 3 13 can be specifically exemplified.
- the above cell line TR— TR ⁇ 3 3 13 is based on the Busyeast Treaty, the Institute of Biotechnology, Industrial Technology Research Institute, Ministry of Economy, Trade and Industry (1-3 1-3 Tsukuba East, Ibaraki, Japan) Deposit No. NIBHFE RM BP—6 873 3 on flight number 3 05—8 5 6 6).
- TR-AST32 As an immortalized astrocyte cell line derived from a transgenic rat into which a large T antigen gene of the SV40 temperature-sensitive mutant tsA58 was introduced, Na + -dependent L- Specific cell lines capable of expressing glutamate transport such as TR-AST32, TR-AST811, TR-AST912, TR-AST932, TR-AST944, etc. Can be exemplified. Based on the Budapest Treaty, the above-mentioned cell line TR—AST 932 was obtained from the Ministry of Economy, Trade and Industry, National Institute of Advanced Industrial Science and Technology, Institute of Biotechnology and Industrial Technology (1-3, Tsukuba, Higashi, Ibaraki, Japan).
- the immortalized cerebral capillary pericyte cell line derived from the transgenic rat into which the large T antigen gene of the temperature-sensitive mutant tsA58 of SV40 has been introduced includes PDGF receptor 3 and angiopoietin.
- Specific examples include cell lines TR-PCT1, TR-PCT2, etc., which have an expression ability of 1. Based on the Budapest Treaty, the above cell line TR—PCT1 was developed by the Ministry of Economy, Trade and Industry,
- the blood-brain barrier reconstruction model in the present invention is a rat-derived immortalized brain hair.
- Corrected form Co-culturing a microvascular endothelial cell line with a rat-derived immortalized astrocyte cell line, or using a rat-derived immortalized brain capillary endothelial cell line and a rat-derived immortalized astrocyte cell line And a rat cell-derived immortalized brain capillary pericyte cell line. Co-culture is performed by culturing these cell lines in contact with each other, or by immortalizing cerebral capillary endothelial cell lines and immortalized astrocyte cell lines, or immortalizing cerebral capillary endothelial cell lines and immortalized cells.
- Non-contact culture of strocite cell line and immortalized cerebral capillary pericyte cell line through a membrane that can penetrate, for example, humoral factors, but not cells This can be done by: By co-culturing in a non-contact state through such a membrane, not only the immortalized cerebral capillary endothelial cell line can be easily separated from other cells, but also a membrane having various pore sizes can be used. Thus, the molecular weight of a humoral factor responsible for signal transmission between cells can be estimated.
- a marker gene of the blood-brain barrier of the present invention for example, an alkaline phosphatase gene, adartamyl transpeptida
- the immortalized cerebral capillary endothelial cell line with enhanced expression of the G1ut1 gene is a transgenic rat into which the large T antigen gene of the temperature-sensitive mutant tsA58 of SV40 has been introduced.
- Immortalized brain capillary endothelial cell line derived from the same. Co-culture of the immortalized astrocyte cell line, or the immortalized astrocyte cell line and the immortalized brain capillary pericyte cell line.
- Immortalized brain capillary endothelial cell lines with enhanced expression of the marker gene of the blood-brain barrier include the alkaline phosphatase gene after co-culture compared to when cultured alone without co-culture.
- the blood-brain barrier remodeling model of the present invention and the immortalized brain capillary endothelial cells with enhanced expression of the blood-brain barrier marker gene of the present invention provide a blood-brain barrier that restricts the transfer of substances from blood to brain tissue. It can be used for research on nutrients and metabolism in the brain, drug permeation into the brain, and defense mechanisms at the blood-brain barrier.
- a blood-brain barrier remodeling model of the present invention and a blood-brain barrier formation promoting or inhibiting substance, and a blood brain using an immortalized brain capillary endothelial cell with enhanced expression of one gene of the blood-brain barrier marker of the present invention A method for screening a substance that promotes or inhibits barrier permeation and a substance that permeates or impregnates the blood-brain barrier is described.
- Screening for a substance that promotes or inhibits blood-brain barrier formation using a blood-brain barrier reconstruction model involves contacting a test substance with an immortalized brain capillary endothelial cell line during or before or after co-culture, and This can be done by measuring the degree of expression of the marker gene and comparing and evaluating it with a control in which the test substance is absent.
- Screening of a substance that promotes or suppresses blood-brain barrier formation involves contacting a test substance with an immortalized brain capillary endothelial cell line with enhanced expression of the blood-brain barrier marker gene, and a marker for the blood-brain barrier marker. It can also be performed by measuring the degree of expression enhancement and comparing and evaluating the control in the absence of the test substance.
- the blood-brain barrier formation-promoting substance obtained by these screenings can be expected as a therapeutic agent due to blood-brain barrier dysfunction, and these blood-brain barrier formation-promoting or suppressing substances are at the cellular level. Useful for studying blood-brain barrier formation.
- blood-brain barrier penetration enhancer or inhibitor using blood-brain barrier reconstruction model
- Screening involves contacting a known blood-brain barrier transmissive substance or a blood-brain barrier impervious substance with a test substance with an immortalized cerebral capillary endothelial cell line during or before or after co-culture, and It can be performed by measuring the degree of penetration of the brain barrier permeating substance or the blood-brain barrier non-permeating substance into the immortalized brain capillary endothelial cells, and comparing and evaluating with the control in the absence of the test substance. .
- Screening for a substance that promotes or inhibits blood-brain barrier penetration involves testing a known substance that is permeable to the blood-brain barrier or a substance that is impervious to the blood-brain barrier, and a test substance, by using Contact with a capillary endothelial cell line to immortalize these blood-brain barrier permeating substances or blood-brain barrier impervious substances.Measure the extent of permeation into brain capillary endothelial cells, and check for the absence of the test substance. It can also be done by comparing and evaluating with the case of.
- the substances that enhance or inhibit the blood-brain barrier permeation obtained by these screens can be used for studies on nutritional metabolism in the brain, drug permeation into the brain, and research on defense mechanisms at the blood-brain barrier.
- the facilitator is useful as a concomitant drug with centrally acting drugs (anti-dementia drugs, drugs for treating brain tumors, drugs for viruses, and drugs for psychiatric nerves).
- Screening of the blood-brain barrier permeating or non-permeating substance using the blood-brain barrier remodeling model immortalizes the test substance by contacting it with the brain capillary endothelial cell line during or before or after co-culture.
- the measurement can be performed by measuring the degree of penetration of the test substance into the brain capillary endothelial cells, and comparing and evaluating the control with the control without the test substance.
- the screening of the blood-brain barrier permeating or non-permeating substance is performed by contacting a test substance with an immortalized brain capillary endothelial cell line in which expression of the blood-brain barrier marker gene is enhanced. It can also be performed by measuring the degree of penetration of the test substance into the cells, and comparing and evaluating this with a control in which the test substance is absent.
- the blood-brain barrier permeable substance obtained by these screenings is
- Transgenic rats into which the DNA of the temperature sensitive mutant tsA58 of SV40 was introduced were prepared by the following procedure.
- genomic DNA of SV40 temperature-sensitive mutant tsA58 was used.
- the genomic DNA of tsA58 was opened with the restriction enzyme BamHI, introduced into the BamHI site of pBR322, and
- the DNA clone pSVtsA58ori (—) was converted from the I sequence to Sac ⁇ and deleted as an ori (—), which deletes the SV40 origin of replication (ori).
- DNA for introduction was prepared according to a conventional method. That is, pSVtsA58ori (I) —2 of plasmid DNA obtained by amplifying a large amount in Escherichia coli was digested with restriction enzyme BamHI (Takara Shuzo), and then agarose was digested. The DNA was separated by electrophoresis (1% gel; manufactured by Boehringer), and the gel was dissolved. The DNA was recovered by phenol-cloth form treatment and ethanol precipitation treatment.
- the recovered purified DNA is dissolved in TE buffer (1 OmM Tris-HC1 containing 1 mM EDTA; pH 7.6) and a solution containing 170 g / m1 purified DNA I got This DNA solution was diluted with an injection buffer (10 mM Tris-HC1 containing 0.1 mM EDTA; pH 7.6) to 5 g Zml, and the DNA solution for injection was diluted. Was prepared. The prepared DNA solution was stored at 120 ° C until injection.
- Microinjection of the above prepared DNA solution for injection into fertilized eggs at the pronuclear stage was performed as follows. Sexually mature 8-week-old chairs are bred in a light-dark cycle for 12 hours (4: 00 to 16: 00 for light hours), at a temperature of 23 ⁇ 2 ° C, and a humidity of 55 ⁇ 5%. Then, the female estrous cycle was observed by a vaginal smear, and the date of hormone treatment was selected. First, 150 IU / kg of pregnant female serum gonadotropin (manufactured by Nippon Zenyaku; PMS
- mice were mated by cohabitation with males. 3 hours after hCG administration, fertilized eggs at pronuclear stage were collected by fallopian tube perfusion Oviduct Perfusion and egg culture were performed using mK RB solution (Toyoda Y. and Chang MC, J. Reprod. Fertil., 36, 9-22, 1974).
- the cerebrum was isolated from the transgenic rat (1 animal) into which the large T antigen gene of the temperature-sensitive mutant tsA58 of SV40 obtained in Example 1 was introduced.
- the cerebrum excised in a clean bench was ice-cooled with an adjustment buffer (10 mM Hepes, lOUZ ml of benzylpenicillin potassium, 100 / igzml of streptomycin sulfate, 0.5% ⁇ serum albumin).
- the obtained pellet was treated with lml of enzyme solution (0.01% collagenase / dispase (Boehrmger Manheim), 100 Um1 of benzylpenicillin potassium, ⁇ 100 ig / ml of streptomycin sulfate, 20 UU / m 1 of deoxyribonuclease I and 0.147 xg of m 1 tosyl-lysine-chloromethylketone).
- enzyme solution 0.01% collagenase / dispase (Boehrmger Manheim)
- 100 Um1 of benzylpenicillin potassium ⁇ 100 ig / ml of streptomycin sulfate, 20 UU / m 1 of deoxyribonuclease I and 0.147 xg of m 1 tosyl-lysine-chloromethylketone.
- Enzyme treatment (37 :, 30 minutes) was performed in the obtained water bath to separate capillaries from unnecessary tissues.
- the pellet was obtained by centrifugation (600 ⁇ g, 5 minutes, 4 ° C.). To remove unnecessary tissue from the resulting pellet, suspend the pellet in 10 ml of HBSS containing 16% dextran, and centrifuge (1, 000 Xg, 15 minutes, 4 hours). ° C) to obtain a pellet of the capillary fraction. The obtained pellet was suspended again in a 1 ml enzyme solution and subjected to enzyme treatment (37 ° C, 30 minutes) to cut the capillaries. The pellet was obtained by centrifugation (600 ⁇ g, 5 minutes, 4 ° C.).
- the obtained pellet was added to 2 ml of a culture solution (15 // g / 1 endothelial cell growth factor, 100 U / m1 of benzylpenicillin potassium, 100 x gZml of streptomycin sulfate). , 2.5 ⁇ g Zm 1 amphotericin B) and dispersed in DMEM), and seeded on one 35 mm ⁇ culture dish (Becton Dickinson) coated with one collagen type I. 3 3 ° C carbon dioxide incubator at (5 C 0 2 - 9 5 % A ir, saturated humidity) and incubated in a (primary culture). The medium was changed twice a week, and the passage was performed using trypsin solution (0.05% Trypsin,
- the cells were detached using 0.5 mM EDTA (manufactured by Gibco BRL) and dispersed and seeded. Passaging was performed at approximately weekly intervals. After three passages, were seeded in 1 0 2 to 1 0 3 cells were coated with collagen type I 1 0 ⁇ ⁇ culture dish one (Becton Dickinson Co., Ltd.). Colonies were formed by culturing in a carbon dioxide incubator at 33 ° C. The medium was changed twice a week, colonies that formed colonies at a relatively high growth rate after 7 to 10 days were isolated from surrounding cells using a vesicinate cup, and the obtained cells were recovered again. 0 mm (/ inoculated in a culture dish and cultured in a carbon dioxide incubator at 33 ° C to form colonies. Using a penicillin cup, colonies with a relatively high growth rate were isolated from surrounding cells. 5 cell lines (TR-BBB)
- TR-BBB13 strain has been deposited under the Budapest Treaty with the Ministry of Economy, Trade and Industry of Japan, as a deposit number FEPMBP-66873 at the Research Institute of Biotechnology and Industrial Technology.
- Example 2-1 Expression of the large T antigen protein in the five cell lines obtained in Example 2-1 above was determined by Western blotting (Experimental Medicine Separate Volume Biomanual UP Series “Cancer Research Protocol by Molecular Biological Approach”). (8-115 pages, Yodosha, published in 1995).
- Five cell lines (passage number: 20) were cultured in a 90 ⁇ culture dish until saturation. The collected cells are solubilized with 3% SDS-PBS (pH 7.4), centrifuged (10,000 rpm for 10 minutes) to remove insoluble fractions, and then subjected to a blood feed method. (Using a protein assay kit II manufactured by BIO-RAD) to quantify the total protein mass.
- Example 2-1 The cell lines obtained in Example 2-1 above were brain capillary endothelial cells, and the expression of GLUT-1 transporter and p-glycoprotein was assayed by Western blotting.
- an anti-macro antibody was used as a primary antibody using a nitrocellulose membrane prepared in the same manner as in Example 2-2.
- Mouse GLUT-1 antibody (Chemicon, Temecular, CA) or anti-P_glycoprotein ⁇ sagi antibody (anti-mdr antibody, Oncogene Research Products3 ⁇ 4S3 ⁇ 4), and HRP-labeled ⁇ mouse IgG antibody (Amersham GLUT-1 protein or p-glycoprotein-specific reaction using the ACL's ECL Western Blotting Detection System (RPN2106M1) or HRP-labeled anti-Egret IgG antibody (Cappel). Detected. Expression of GLUT-1 protein and p-glycoprotein was confirmed in all five cell lines. Therefore, the obtained five types of cells were identified as brain capillary endothelial cells.
- the cell lines TR_BBB1, TR—BBB5, TRBBB6, TRBBB11, and TRBBB13 obtained in Example 21 described above had a functional GLUT-1 transport carrier.
- the ability to take up (3-o-methyl-D-glucose) was measured, and it was confirmed that it had a functional GLUT-1 transporter by showing a concentration-dependent glucose transport ability. That is, the TRBBB strain was seeded on a 24-well cell culture plate at a concentration of 3 XI 0 5 Z ⁇ Zml, and cultured for 24 hours in a 33 ° C carbon dioxide gas culture medium to make the cells confluent. .
- the measurement of the uptake of 3- 3MG was performed as follows. First, after removing the medium was aspirated, was 0.
- the same operation was performed for 0 minute and 1 minute.
- the cells were solubilized in 1 ml of PBS containing 1% Triton X-100 in lm 1 and the radioactivity was measured using a liquid scintillation counter to confirm the linearity of 3 — OMG uptake. did. As a result, a capture time of 20 seconds was set.
- Example 2-1 It was measured according to a standard method that the cell line obtained in the above Example 2-1 expresses the activity of lipophilic phosphatase and the activity of aldaramil transbeptidase expressed in brain capillary endothelial cells. .
- Alkaline phospha B-test and A-1 GTP-test (manufactured by Wako Pure Chemical Industries, Ltd.) were used for the measurement according to the standard measurement method described in each kit.
- the amount of protein was measured by the Bradford method (Protein Atsushi Kit II; manufactured by BIO-RAD).
- the alkaline phosphatase activity and the aardal mil transpeptidase activity were 8.7 to 25.8% and 5, respectively, based on the rat brain capillary rich fraction (Brain Capillaries). It showed 4 to 22.6%, indicating expression of brain capillary endothelial cell-specific enzyme. Table 2 shows the results.
- ALP activity T-G ⁇ _ ⁇ activity cells ⁇ U / mg prote inn (control ratio U / mg protein (control ratio%)
- TR-BBB1 23.7 ⁇ 7.17 (25.83 ⁇ 4) 3.62 ⁇ 0.47 (12.43 ⁇ 4)
- TR-BBB6 22.3 ⁇ 8.78 (24.33 ⁇ 4) 1.58 Sat 0.52 (5.43 ⁇ 4)
- the cerebrum was isolated from the transgenic rats (5) into which the large T antigen gene of the temperature-sensitive mutant tsA58 of SV40 obtained in Example 1 was introduced. Separation and recovery of brain astrocyte cells were performed using the enzymatic method as follows. Cerebral sterilized at 1 2 1 ° C, 1 5 min, separation bar Ffa (1 2 2 mM of N a C l, 3 mM of KC 1, 1. 4 mM of C a C l 2, 1. 2 mM of M g S 0 4, 0. 4mM of K 2 HP 0 4, 1 0 m M of G lucose, l O mM of H epes; p H 7.
- the obtained cell suspension was cultured in a 5% C ⁇ 2 incubator at 37 ° C for 2 days in a culture dish (Cornmg; # 43016). On day 3 3 3 ° and transferred to 5% C 0 2 incubator C and the culture was continued.
- the cells were treated with a 0.1% collagenase Z dispase solution at 37 ° C for 5 minutes, and then left at 4 ° C for 2 hours to detach the cells from the culture dish.
- Cloning of the cells was performed by the colony forming method. 1-3 times have passaged cells Nitsu, after peeling off the cells were plated 1 0 2 to 1 0 3 cells into 9 0 mm culture dish, were colony formation.
- TR-AST32, TR-AST811 and TR-AST912 TR-AST933 and TR-AST943 strains were named TR-AST32, TR-AST811 and TR-AST912 TR-AST933 and TR-AST943 strains, respectively.
- TR-AST932 strain was deposited under the Budapest Treaty with the Nippon International Trade and Industry Institute of Industrial Science and Technology, Institute of Biotechnology and Industrial Technology under the accession number F ⁇ ⁇ ⁇ ⁇ -62883. Have been.
- glial fibrillary acidic protein a protein
- PLP a mixture of sodium perchlorate, L-lysine hydrochloride and paraformaldehyde
- Blocking was performed for 60 minutes at room temperature using the locking solution. After incubating the anti-GFAP colonies diluted 100 times at room temperature for 60 minutes, the cells were washed three times with rinse buffer, and the peroxidase-labeled anti-rabbit IgG diluted 1: 500 was acted on for 60 minutes. I let it. After thoroughly washing the sample with a rinse buffer, a color reaction was performed. An ice-cooled coloring solution was added, and the mixture was observed under a microscope. When the sample developed color, the reaction was stopped by adding cold PBS. At this time, the intensity of the coloring was compared with that of the negative control. A 3,3-diam inobenzidine-PBS solution containing 30% hydrogen peroxide was used as a coloring reagent.
- TR-AST32, TR-AST811 and TR-AST912, TR-AST932 and TR_AST943 strains there was a request to show the expression of GFAP. Staining was confirmed. Therefore, it was confirmed that TR-1 AST32, TR-AST811, TR-AST910, TR-AST932, and TR-AST944 were strains of fast mouth cell lines. Was.
- the medium was aspirated, washed with the above uptake buffer II warmed to 37 ° C, and then heated to 37 ° C 4 6.
- the tracer solution was removed, and the plate was washed three times with 1 ml of uptake buffer II at 4 ° C. 1% of 0.75 m 1
- L- G lu uptake is concentration-dependent, the Michaelis constant (Km) is 9 6 M, the maximum uptake rate constant (Vm ax) is 1. It was 8 nmol Zmin / mg protein. This uptake was significantly inhibited by Na +-freebuffer, and was significantly inhibited by other substrates such as L-asparticacid and D-asparticacid. Table 3 shows the results. From Table 3, it was confirmed that the obtained cell line retained the original function of astrocyte.
- the pellet obtained by centrifuging the chiller (4500 xg at 4 ° C for 15 minutes) was added to 2.4 ml of the enzyme solution [0.066% collagenase / dispase (Boehringer Suspended in PBS containing 0.033% of 88 (manufactured by Sigma)], and subjected to enzyme treatment (37 ° C, 3 hours) in an aqueous solution with shaking.
- the extracellular matrix was separated and centrifuged (600 xg at 4 ° C for 5 minutes) to obtain a pellet.
- the obtained pellet was added to a 10 ml culture medium (100 / m1 benzylpenicillin potassium, 100 / gzm1 streDtomycin sulfate 2.5.50 / ig Zm1 ampnotericm B, 20% FCS Was dispersed in DMEM) and seeded on four 35 mm ⁇ i) culture dishes (Falcon). 3 3 ° C CO 2 incubator (5% of the C_ ⁇ 2 _ 95% of the A ir, saturated humidity) and incubated in a (primary culture). The medium was changed twice a week, and subculture was performed at approximately one week intervals using a trypsin solution (0.05% Trypsin, 0.53 mM EDTA; Gibco BRL).
- TR-PCT1 1 TR — PCT strain
- TR-PCT2 2 9 Colonies were isolated from surrounding cells to obtain two cell lines (TR-PCT1, TR-PCT2).
- TR-PCT1 one TR — PCT strain has been deposited with the Nippon International Trade and Industry Institute of Industrial Science and Technology, Institute of Biotechnology, Industrial Technology under the Busyeast Treaty under the accession number FEPMBP — 7204. . 4-2 (Confirmation of large T antigen protein)
- Example 4 Large T antigen proteins of the two cell lines obtained in 11 were detected by the Western plot method described in Example 2-2. That is, each of the two cell lines was washed with PBS, and then solubilized with 1 mL of a solubilization solution (1% DS, 10 mM Tris, ImM EDTA, and 10% glycerin). After heating at 100 ° C for 10 minutes, centrifugation (100 rpm at 10 minutes) was performed to remove insoluble fractions, and then the Bradford method (BCA protein assay reagent A manufactured by PIERCE) was used. Used) to determine the total protein content. After 10 / xg of each protein was separated by SDS polyacrylamide gel electrophoresis, it was transferred to a nitrocellulose membrane.
- a solubilization solution 1% DS, 10 mM Tris, ImM EDTA, and 10% glycerin. After heating at 100 ° C for 10 minutes, centrifugation (100 rpm at 10 minutes) was performed to
- Anti-SV40 large T antigen mouse antibody (DP02-C; manufactured by CALBIOCHEM) was used as the primary antibody on the nitrocellulose membrane blocked with 3% skim milk solution, and HRP-labeled anti-mouse IgG was used as the secondary antibody.
- An antibody (Amersham) was allowed to react, and a large T antigen-specific reaction was detected using an ECL Wessling detection system (RPN210M1) manufactured by Amersham. Table 6 shows the results. As a result, large T antigen protein was confirmed in all of the obtained two cell lines.
- the obtained cell line was cultured in a monolayer, immunostained for PDGF receptor) 3 and Thy-1 expressed on the cell membrane, and confirmed using a microscope.
- Example 4 The cell line T R —PCT 1 obtained in 11 was cultured on a cover glass of a 24-well dish (Falcon). After removing the culture medium and washing the cells with PBS,
- the cell line obtained in Example 4-11 was cultured in a monolayer, and angiopoetin-11, osteopontin, and ICAM-1 expressed in the cells were detected by RT-PCR. That is, the cell line TR-PCT1 was cultured in a 100 mm ⁇ culture dish (Falcon). After removing the culture solution and washing the cells with PBS, the cells were collected by a cell scraper (manufactured by IWAKI), and total RNA was extracted with an RNA extraction reagent (Trizol; manufactured by Gibco). Using reverse transcriptase (Rev Tra Ace: manufactured by TOYOBO), cDNA was synthesized from all the extracted RNAs, and expressed by PCR (exTacj; manufactured by Takara Shuzo). It was confirmed. Confirmation of the PCR amplification product was performed by electrophoresis on a 5% acrylamide gel. As a result, the cell line T R—PC T 1
- Example 4-11 (Confirmation of calcium deposit in matrix by high-density culture)
- the cell line obtained in Example 4-11 was cultured in a monolayer, and calcium deposited in the matrix was confirmed by Vonkossa staining. . That is, 1 0 6 cell lines TR - the PCT 1, (manufactured by Falcon) 1 0 0 mm phi cultured catcher Ichire in DMEM and 1 0 O mm c) I collagen coated culture Shah Ichire (IWAKI (Manufactured by the company). 1 OmM 3-phosphoric acid phosphate was added to the culture solution of the ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ type collagen-coated culture dish.
- TR_BBB 13 obtained by the above Example 2 was added to DMEM 1 [DMEM (manufactured by Nissi; # 05915) # 105915] to give a final concentration of 10% fetal serum (manufactured by JRH; # 1203-78 P), 100 UZm1 penicillin, 100 / xg Zm1 streptomycin, 2 mM glutamine (GIBCO; # 10378-8) 16), 15 // g / ml of bECGF (manufactured by Behringer Inc .; # 103333484)], and a collagen type I-coated culture dish (manufactured by IWAKI) ; # 4 0 2 0 - 1 0) were seeded in advance cultured under conditions of 5% C 0 2 Z 9 5 % AIR at 3 3 ° C.
- TR-AST932 obtained in Example 3 and TR-PCT1 obtained in Example 4 were converted to DMEM 2 [DMEM (Nissi; # 05915)].
- 10% fetal serum at final concentration (manufactured by JRH; # 1203 — 78P), lOO UZml penicillin, lOOg Zm1 streptomycin, 2 mM glue evening Min (GIBC_ ⁇ Ltd .; # 1 0 3 7 8 - 0 1 6)] that contains the culture dish; in (Corning Inc. # 4 3 0 1 6 7) , 5% C 0 2 Z 9 5% AIR
- the cells were pre-cultured at 33 ° C under the following conditions.
- Millicell 3.0 p.m c u l t u r e i n s e r t, 30 mm d i a m e t e r, # PIT P 0 3 0 5 0)
- Reagent A and reagent B of BCA enzyme assay reagent were mixed at a ratio of 50: 1 and left for 1 day to prepare a working solution (working solution). solution).
- a working solution working solution
- BCAProtein Assay Reagent manufactured by PIRCE: # 531-2072
- To 0.1 ml of each of the above three types of cultured samples (control, A + endothelial cells, A + P + endothelial cells) and BSA standard solution add 2 ml of ⁇ -King solution, and add After incubation with C for 30 minutes and cooling to room temperature, the absorbance at a wavelength of 562 nm was measured.
- the protein concentration of each of the three samples was calculated based on a calibration curve prepared using the BSA standard solution.
- the absorbance of these solutions was measured at a wavelength of 405 nm, and the ALP activity value (mUZm 1) corresponding to the absorbance was calculated from a calibration curve prepared in advance, and the protein concentration per 1 mg of protein was calculated from the above protein concentration. ALP activity value was determined. The result is shown in figure 2.
- the ALP activity value was 3.93 ⁇ 0.39 mUZmg in the control cultured only with the brain capillary endothelial cell line (TR-BBB13), and the Astrocyte cell line (TR — A brain capillary endothelial cell line (A + endothelial cell) co-cultured with AST 932) was 23.5 ⁇ 4.69 mU / mg, and a brain capillary pericyte cell line (TR-PCT 1 ) And 25.0 ⁇ 1.6 mUZmg in the brain capillary endothelial cell line ( ⁇ + ⁇ + endothelial cells) co-cultured with the first mouth site cell line.
- the ⁇ -GTP activity values were 4.13 ⁇ 0.68 mU / mg for control, 18.5 ⁇ 1.6 mU / mg for A + endothelial cells, and A + P +, respectively. In endothelial cells, it was 10.3 ⁇ 0.5 ⁇ 5 mUZmg. From these facts, the co-culture with TR-AST932 increased the ALP activity by a factor of 6 and increased the GTP activity by a factor of 4.5 compared to the TR-BBB13 alone culture. Was found to increase. In addition, ALP activity of the brain capillary endothelial cell line co-cultured with TR—PCT1 and TR—AST932 increased 6-fold.
- G3PDH (housekeeping gene) in the culture of the above-mentioned brain capillary endothelial cell line alone (control) and in the brain capillary endothelial cell line (A + endothelial cell) co-cultured with the astrocyte cell line ) And GLUT-1 (glucose transport 1) were detected by semi-quantitative RT-PCR.
- the above-mentioned brain capillary endothelial cell line was cultured in Millicell (Millipore; Millicell: 3.0 um culture insert, 30 mm diameter, # PITP350).
- RNA extraction reagent Trizol; manufactured by Gibco.
- RevTra Ace Toyobo
- G3PDH [G3PDH-F: 5′-ACC AC AGT C CAT GC CAT CAC—3 ′ (SEQ ID NO: 3), G3PDH-R : 5 '— TCCAC CAC CC TGTT GCT GTA-3' (SEQ ID NO: 4)], GL UT-1 [GL UT-1 1 F: 5 'one GATGAT GAAC C TGTT GGCCT-3' (SEQ ID NO: 5), GL UT—1—R: 5 ′ 1 AG C GGAA C AG CTC CAAGATG-3 ′ (SEQ ID NO: 6)] was used, respectively. 7-3 (PCR)
- the rExTaq was added to each of the above-mentioned primers of 0.21 and 2, and a PCR reaction was carried out with a total amount of 50 zl.
- the thermal cycle program consists of a first denaturation at 94 ° C for 3 minutes, followed by heat denaturation at 94 ° C for 1 minute, extension at 57 ° C for 1 minute, and annealing at 72 for 1 minute. Was repeated 25 times, and finally annealing was performed at 72 ° C. for 10 minutes. Thereafter, the PCR amplification product was separated by agarose gel (2%) electrophoresis, and then stained with ethidium bromide, and the intensity of each band was measured using a CCD image analyzer. Fig. 4 shows the results.
- the present invention it is possible to reconstruct an in vivo blood-brain barrier experiment system that is closer to in vivo, and a co-culture system using a conditional immortalized cell line is dramatically more cell-efficient than conventional co-culture. It has been shown that cell-cell interactions are promoted and the blood-brain barrier can be reproduced to a point close to its original function. Therefore, by using the blood-brain barrier reconstruction model and the like of the present invention, not only basic research results on the blood-brain barrier can be obtained, but also centrally acting drugs (anti-dementia drugs) based on the blood-brain permeation mechanism of drugs. , Brain tumor drugs, virus drugs, neuropsychiatric drugs) and drugs that cause central side effects
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Abstract
A blood-brain barrier reconstruction model which is highly useful in fundamental studies on the blood-brain barrier permeation mechanism, and screening drugs with central action based on the blood-brain barrier permeation mechanism, drugs suffering from problems concerning side effect on the center, drugs targeting various receptors expressed in cerebral capillary endothelial cells or drugs targeting disorders in cerebral capillary endothelial cells. This blood-brain barrier reconstruction model is prepared by cocultivating an immortalized cerebral capillary endothelial cell line originating in a transgenic rat having large T antigen gene of a temperature-sensitive mutant SV 40 tsA58 transferred thereinto with an immortalized astrocyte cell line originating in the same rat in a non-contact state, or cocultivating the above-described immortalized cerebral capillary endothelial cell line and the immortalized astrocyte cell line together with an immortalized capillary adventitial cell line originating in the same rat in a non-contact state.
Description
共培養による血液脳関門再構築モデル 技術分野 Blood-brain barrier reconstruction model by co-culture
本発明は、 血液脳関門再構築モデル、 より詳しくは、 ラッ ト由来の不 死化脳毛細血管内皮細胞株、 不死化ァス トロサイ ト細胞株、 不死化脳毛 細血管周皮細胞株を共培養する明ことによる血液脳関門再構築モデルの作 製方法や、 共培養することにより得田られる血液脳関門のマーカー遺伝子 の発現が増強した不死化脳毛細血管内皮細胞株や、 かかる血液脳関門モ デルや血液脳関門のマーカー遺伝子の発現が増強した不死化脳毛細血管 内皮細胞株を用いた血液脳関門形成促進物質等のスク リーニング方法等 に関する。 背景技術 The present invention relates to a blood-brain barrier remodeling model, more specifically, a rat-derived immortalized brain capillary endothelial cell line, an immortalized astrocyte cell line, and an immortalized brain capillary pericyte cell line. A method for creating a blood-brain barrier remodeling model by culturing, an immortalized brain capillary endothelial cell line with enhanced expression of a blood-brain barrier marker gene obtained by co-culture, and such a blood-brain barrier The present invention relates to a method of screening a substance promoting blood-brain barrier formation using an immortalized brain capillary endothelial cell line with enhanced expression of a model or a marker gene of the blood-brain barrier. Background art
血液脳関門 （blood-bram b arrier) とは、 血液から脳組織内への物質 の移行を制限する関門であり、 これによつて、 脳は有害物質などから守 られている。 ニコチン、 カフェイン、 ヘロイン等の脂溶性物質はこの血 液脳関門を容易に通過できるが、 一般に極性物質、 強電解質など非脂溶 性物質は通過しにくいものの、 脳の代謝に必要な D —グルコース等の水 溶性物質はキヤリヤーによって血液脳関門を透過して脳組織へ運ばれる ことが知られている。 脳毛細血管においては、 隣接する内皮細胞同士が タイ ト · ジャンクショ ンと呼ばれる緊密な結合を形成し、 細胞間の隙間 から漏出しないようになっており、 そのため、 脳内に出入りする物質は 原則として脳毛細血管内皮細胞を通過しなくてはならないとされている, 上記のように、 脳毛細血管内皮細胞は、 脳への栄養物質だけでなく細
胞膜に発現している種々の輸送系によって薬物を脳内へ輸送する。 ある いは、 その実体はほとんど解明されていないが、 血液脳関門には脳から 循環血液方向へ神経伝達物質の代謝物および異物を排出する輸送系が働 く特殊な生理機能が存在する。 異物である薬物の中には脂溶性が高いに も係わらず脳への移行性が悪いものも多いことが知られており、 その原 因として血液脳関門に存在する排出輸送系によって一旦脳内へ移行した 薬物がその移行速度の数十倍から数百倍も高い速度で脳から排出される ことが考えられている。 例えばエイズウィルス治療薬であるアジドチミ ジンは血液脳関門排出輸送系の働きによって脳への移行性が著しく制限 されている ( J. Pharmacol. Exp . Ther. , 281 , 369-375 , 1997 , ibid 282, 1509- 1517, 1997)。 このように中枢作用型薬物の脳への移行において血 液脳関門は極めて重要な役割を果たしている。 The blood-brain barrier is a barrier that restricts the transfer of substances from the blood into brain tissue, thereby protecting the brain from harmful substances. Lipid-soluble substances such as nicotine, caffeine, and heroin can easily pass through the blood-brain barrier, but generally non-lipid-soluble substances such as polar substances and strong electrolytes are difficult to pass, but are required for brain metabolism. It is known that water-soluble substances such as glucose permeate the blood-brain barrier and are transported to brain tissue by carriers. In the brain capillaries, adjacent endothelial cells form tight junctions called tight junctions and do not leak out from the gaps between cells, so substances that enter and exit the brain are in principle It has to pass through brain capillary endothelial cells, as mentioned above, brain capillary endothelial cells are not only nutrients to the brain but also fine Drugs are transported into the brain by various transport systems expressed in the alveolar membrane. Or, although its substance is largely unknown, the blood-brain barrier has a special physiological function in which a transport system that excretes metabolites and foreign substances of neurotransmitters from the brain to the circulating blood system works. It is known that many foreign substances, which have high fat-solubility but are poorly migratory to the brain, are known to have been caused by the efflux transport system existing at the blood-brain barrier. It is thought that a drug that has migrated to the brain is excreted from the brain at a rate that is tens to hundreds of times faster than its migration rate. For example, azidothymidine, a drug for treating AIDS virus, is significantly restricted from being transported to the brain by the action of the blood-brain barrier efflux system (J. Pharmacol. Exp. Ther., 281, 369-375, 1997, ibid 282). , 1509-1517, 1997). Thus, the blood-brain barrier plays a very important role in the transfer of centrally acting drugs to the brain.
従来、 血液脳関門のインビトロ実験系としては、 初代培養脳毛細血管 内皮細胞単離方法 (Audus c& Borchardt ら、 Pharm. Res. , 3, 81 -87, 1986) に基づいて、 ゥシから調製して実験が行われてきた。 しかし、 初代培養 細胞では、 例えば中性アミノ酸輸送系で運ばれる L 一 ドーパやへキソ一 ス輸送系で運ばれるグルコースの輸送速度がィンビボ系に比べて数十倍 以下と低いことが報告され （Pardridge ら、 J. Pharmacol. Exp . Ther. , 253, 884-891 , 1990)、 逆に単純拡散による物質透過がィンビボ系よりも 1 5 0倍以上高く、 血液脳関門のモデルとしては不十分であった。 また、 株化脳毛細血管内皮細胞である M B E C— 4 ( Tatsuta T ら、 J. Biol. Chem . , 267 , 20383-20391， 1992) では、 インビボで発現してレ る m d r ( multi drug resistance) 1 a遺伝子産物が発現しておらず、 またイン ビボで発現していない m d r 1 b遺伝子産物が発現している点で正常な 脳毛細血管内皮細胞ではないと考えられている。 Conventionally, an in vitro experimental system for the blood-brain barrier has been prepared from yeast based on the primary cultured brain capillary endothelial cell isolation method (Audusc & Borchardt et al., Pharm. Res., 3, 81-87, 1986). Experiments have been done. However, in primary cultured cells, for example, it has been reported that the transport rate of glucose transported by the L-dopa or hexose transport system, which is transported by the neutral amino acid transport system, is several tens of times lower than that of the in vivo system ( Pardridge et al., J. Pharmacol. Exp. Ther., 253, 884-891, 1990) Conversely, the permeation by simple diffusion is more than 150 times higher than that of the in vivo system, and is not a sufficient model for the blood-brain barrier. there were. In addition, MBEC-4 (Tatsuta T et al., J. Biol. Chem., 267, 20383-20391, 1992), a established brain capillary endothelial cell, shows that mdr (multi drug resistance) 1 is expressed in vivo. a It is considered that it is not a normal brain capillary endothelial cell in that the gene product is not expressed and the mdr1b gene product that is not expressed in vivo is expressed.
一方、 脳毛細血管内皮細胞はァス トロサイ ト細胞との共培養により、
脳毛細血管内皮細胞の G L UT— 1発現能力を高めることが知られてい る （Hayashi Y.ら， GLIA, 19, 13-26, 1997)。 しかし、 共培養に使用され るァス ト口サイ ト細胞は初代培養ァス トロサイ ト細胞であるために、 毎 回調製する必要があるうえ、 通常生後 1 日ラッ 卜から分離培養している ために成熟ラッ トのァス トロサイ ト細胞とは異なる （Swanson R. A.ら， J Neurosci., 17, 932-940, 1997) という問題がある。 これまで、 ァス ト 口サイ ト細胞株として、 KG— 1 — C、 U 2 5 1、 G I — 1等のヒ ト由 来の神経膠腫瘍細胞 （グリア細胞） や R C R— 1、 C 6等のラッ ト由来 腫瘍化グリア細胞が使用されてきたが、 これらグリア細胞は G F A P (glial fibrillary acidic protein) を発現し、 ァス トロサイ トの性質を有 しているものの、 ミエリ ン鞘を形成するオリゴデンドロサイ トの性質も 共に有しているため、 本来のァス トロサイ トだけの機能を反映していな い。 例えば、 C 6細胞ではァス トロサイ 卜が本来有している N a +依存 性 L _グルタミン酸トランスポー夕一である G L UT— 1及び G L A S Tが発現しておらず、 神経細胞に発現する蛋白質である E AA C 1が発 現してレ る (PalosT. P.ら， Mol. Brain Res., 37, 297-303, 1996)。 On the other hand, brain capillary endothelial cells are co-cultured with astrocyte cells, It is known to enhance the ability of brain capillary endothelial cells to express GLUT-1 (Hayashi Y. et al., GLIA, 19, 13-26, 1997). However, since the astrocyte site cells used for co-culture are primary cultured astrocyte sites, they need to be prepared every time, and they are usually separated and cultured from rats one day after birth. However, there is a problem that it is different from mature rat astrocyte cells (Swanson RA et al., J Neurosci., 17, 932-940, 1997). To date, the first oral site cell lines include human-derived glial tumor cells (glial cells) such as KG-1—C, U251, and GI-1 and RCR-1 and C6. Although neoplastic glial cells derived from rat have been used, these glial cells express GFAP (glial fibrillary acidic protein) and have the properties of astrocyte, but oligos that form myelin sheath Since it also has the properties of dendrosite, it does not reflect the functions of the original astrocyte only. For example, C6 cells do not express GLUT-1 and GLAST, which are native Na + -dependent L_glutamic acid transporters in astrocytes, and are proteins expressed in nerve cells. A certain EAACl is expressed (PalosT. P. et al., Mol. Brain Res., 37, 297-303, 1996).
脳毛細血管内皮細胞は、 その周りが周皮細胞及びァス トロサイ ト細胞 で覆われていることが報告されている （Sci. Am.255, 74-83， 1886)。 ま た、 林らの報告 （Hayashi, Y.ら， GLIA 19， 13-26, 1997) によると、 ラ ッ ト胎児のァス ト ロサイ ト細胞と ヒ ト臍帯静脈内皮細胞 （ human umbilical cord vein) の共培養により、 ヒ ト臍帯静脈内皮細胞単培養と 比較して、 ァ ー G T P活性が 5倍高くなり、 G L UT— 1 の mRNAが 9〜 1 1. 6倍上昇したことから、 ァス トロサイ ト細胞が脳毛細血管内 皮細胞とクロス トークして血液脳関門を形成していると考えられている ( しかし、 この報告によると、 ラッ ト胎児由来のァス トロサイ ト細胞と、 ヒ ト臍帯静脈由来の内皮細胞とを用いており、 血液脳関門再構築モデル
とは言い難く、 かつ調製が困難である。 It has been reported that brain capillary endothelial cells are surrounded by pericytes and astrocyte cells (Sci. Am. 255, 74-83, 1886). According to Hayashi et al. (Hayashi, Y. et al., GLIA 19, 13-26, 1997), rat fetal astrocytes and human umbilical cord vein endothelial cells were used. Coculture with human umbilical vein endothelial cells resulted in a 5-fold increase in GTP activity and a 9- to 1.1.6-fold increase in GLUT-1 mRNA. Cells are thought to cross-talk with brain capsular endothelial cells to form the blood-brain barrier ( although, according to this report, rat fetal astrocyte cells and human umbilical cord) Blood-brain barrier reconstruction model using vein-derived endothelial cells And it is difficult to prepare.
また、 本発明者らによる特開平 1 1 — 3 4 1 9 8 2号公報には、 温度 感受性変異株 S V 4 0 t s A 5 8のラージ T抗原遺伝子を導入した トラ ンスジエニックマウスより樹立した脳毛細血管内皮細胞株と、 温度感受 性変異株 S V 4 0 t s A 5 8のラージ T抗原遺伝子を導入したトランス ジエニックラッ 卜より樹立したァス トロサイ ト細胞株とを共培養するこ とにより、 脳毛細血管内皮細胞の G L U T— 1発現能力を高まることが 開示されている。 しかし、 共培養に用いた脳毛細血管内皮細胞株とァス トロサイ ト細胞株とは、 由来する動物種を異にすることから、 厳密な意 味での最適血液脳関門再構築モデルとするにはなお改良の余地があった, 血液脳関門は薬物輸送や脳代謝機能などにおいて重要な役割を果たし ており、 現在までにインビト口での再構築モデルが種々提案されている 力 厳密な意味での最適血液脳関門再構築モデルを作製することは困難 とされてきた。 本発明の課題は、 血液脳関門透過機構に基づいた中枢作 用型薬物 （抗痴呆薬、 脳腫瘍治療薬、 ウィルス治療薬、 精神神経作用薬） 中枢での副作用が問題となる薬物、 脳毛細血管内皮細胞に発現する種々 の受容体を標的とした薬物 （脳微小循環改善薬、 脳浮腫治療薬）、 または 脳毛細血管内皮細胞の障害 （脳血管障害性痴呆症やアルツハイマー型痴 呆症） を標的とした薬物をスク リーニングする上で極めて有用な血液脳 関門再構築モデルを提供することにある。 発明の開示 In addition, Japanese Patent Application Laid-Open No. 11-314982 by the present inventors has established a transgenic mouse into which the large T antigen gene of the temperature-sensitive mutant SV40tsA58 was introduced. By co-culturing the brain capillary endothelial cell line with an astrocyte cell line established from a transgenic rat into which the large T antigen gene of the temperature-sensitive mutant SV40tsA58 was introduced, It is disclosed that the ability of capillary endothelial cells to express GLUT-1 is enhanced. However, since the brain capillary endothelial cell line and the astrocyte cell line used for co-culture differ in the animal species from which they are derived, they must be used in a strict sense as an optimal blood-brain barrier reconstruction model in a strict sense. There is still room for improvement.The blood-brain barrier plays an important role in drug transport and brain metabolism, and various reconstruction models at the in-vitro mouth have been proposed to date. It has been considered difficult to create an optimal blood-brain barrier reconstruction model. An object of the present invention is to provide a centrally acting drug based on the blood-brain barrier permeation mechanism (anti-dementia drug, therapeutic drug for brain tumor, therapeutic drug for virus, psychotropic agent) Drugs targeting various receptors expressed on endothelial cells (cerebral microcirculation improving drug, therapeutic agent for cerebral edema) or disorders of brain capillary endothelial cells (cerebrovascular dementia and Alzheimer's dementia) An object of the present invention is to provide a blood-brain barrier reconstruction model that is extremely useful for screening targeted drugs. Disclosure of the invention
本発明者らは、 上記課題を解決するために鋭意研究し、 同種の動物よ り樹立した不死化細胞株であるラッ ト脳毛細血管内皮細胞株 T R - B B B 1 3 ( F E P M B P— 6 8 7 3 )、 ラッ トァス トロサイ ト細胞株 T R 一 A S T 9 3 2 ( F E P M B P— 6 2 8 3 )、 ラッ ト脳毛細血管周皮細
胞株 T R— P C T 1 ( F E RM B P— 7 0 2 4 ) を用いて共培養する ことにより、 最適血液脳関門再構築モデルを作製することができること を見い出し、 本発明を完成するに至った。 The present inventors have conducted intensive studies to solve the above-mentioned problems, and have developed a rat brain capillary endothelial cell line TR-BBB13 (FEPMBP-68783), which is an immortalized cell line established from animals of the same species. ), Rat trastocyte cell line TR-AST9332 (FEPMBP-62283), rat brain capillaries The present inventors have found that an optimal blood-brain barrier remodeling model can be prepared by co-culturing with the cell line TR-PCT1 (FERMBP-7024), thereby completing the present invention.
すなわち本発明は、 ラッ ト由来の不死化脳毛細血管内皮細胞株と、 ラ ッ ト由来の不死化ァス トロサイ ト細胞株とを共培養することを特徴とす る血液脳関門再構築モデルの作製方法 （請求項 1 ) や、 ラッ ト由来の不 死化脳毛細血管内皮細胞株と、 ラッ ト由来の不死化ァス トロサイ ト細胞 株と、 ラッ ト由来の不死化脳毛細血管周皮細胞株とを共培養することを 特徴とする血液脳関門再構築モデルの作製方法 （請求項 2 ) や、 ラッ ト 由来の不死化脳毛細血管内皮細胞株が、 温度感受性変異株 S V 4 0 t s A 5 8のラージ T抗原遺伝子を導入した トランスジエニックラッ ト由来 の不死化脳毛細血管内皮細胞株であることを特徴とする請求項 1又は 2 記載の血液脳関門再構築モデルの作製方法 （請求項 3 ) や、 温度感受性 変異株 S V 4 0 t s A 5 8のラージ T抗原遺伝子を導入したトランスジ エニックラッ ト由来の不死化脳毛細血管内皮細胞株が、 T R— B B B 1 3 (F E P M B P— 6 8 7 3 ) であることを特徴とする請求項 3記載 の血液脳関門再構築モデルの作製方法 （請求項 4 ) や、 ラッ ト由来の不 死化ァス トロサイ ト細胞株が、 温度感受性変異株 S V 4 0 t s A 5 8の ラージ T抗原遺伝子を導入したトランスジエニックラッ ト由来の不死化 ァス トロサイ ト細胞株であることを特徴とする請求項 1又は 2記載の血 液脳関門再構築モデルの作製方法 （請求項 5 ) や、 温度感受性変異株 S V 4 0 t s A 5 8のラージ T抗原遺伝子を導入したトランスジエニック ラッ ト由来の不死化ァス トロサイ ト細胞株が、 T R— A S T 9 3 2 ( F E P M B P— 6 2 8 3 ) であることを特徴とする請求項 5記載の血液 脳関門再構築モデルの作製方法 （請求項 6 ) や、 ラッ ト由来の不死化脳 毛細血管周皮細胞株が、 温度感受性変異株 S V 4 0 t S A 5 8のラージ
T抗原遺伝子を導入した トランスジエニックラッ ト由来の不死化脳毛細 血管周皮細胞株であることを特徴とする請求項 2記載の血液脳関門再構 築モデルの作製方法 （請求項 7 ) や、 温度感受性変異株 S V 4 0 t s A 5 8のラージ T抗原遺伝子を導入した トランスジェニックラッ ト由来の 不死化脳毛細血管周皮細胞株が、 T R— P C T 1 ( F E R M B P— 7 0 2 4 ) であることを特徴とする請求項 7記載の血液脳関門再構築モデ ルの作製方法 （請求項 8 ) や、 共培養が、 不死化脳毛細血管内皮細胞株 と不死化ァス ト口サイ ト細胞株とを非接触状態で、 又は不死化脳毛細血 管内皮細胞株と不死化ァス ト口サイ ト細胞株及び不死化脳毛細血管周皮 細胞株とを非接触状態で培養する、 非接触系共培養であることを特徴と する請求項 1 ~ 8のいずれか記載の血液脳関門再構築モデルの作製方法 (請求項 9 ) に関する。 That is, the present invention provides a blood-brain barrier remodeling model characterized by co-culturing a rat-derived immortalized brain capillary endothelial cell line and a rat-derived immortalized astrocyte cell line. Preparation method (Claim 1), an immortalized brain capillary endothelial cell line derived from a rat, an immortalized astrocyte cell line derived from a rat, and an immortalized brain capillary pericyte cell derived from a rat A method for preparing a blood-brain barrier remodeling model characterized by co-culturing with a strain (Claim 2), and an immortalized brain capillary endothelial cell line derived from a rat is a temperature-sensitive mutant SV40tsA. 3. The method for preparing a blood-brain barrier remodeling model according to claim 1 or 2, which is an immortalized brain capillary endothelial cell line derived from a transgenic rat into which a large T antigen gene has been introduced. Item 3) and the temperature-sensitive mutant SV 40 ts A58 The blood-brain barrier according to claim 3, wherein the immortalized brain capillary endothelial cell line derived from the transgenic rat into which the large T antigen gene has been introduced is TR-BBB13 (FEPMBP-6873). A method for preparing a reconstructed model (Claim 4), and a method in which a rat-derived immortalized astrocyte cell line is transgenic into which a large T antigen gene of a temperature-sensitive mutant SV40tsA58 was introduced. 3. The method for preparing a blood-brain barrier remodeling model according to claim 1 or 2, which is an immortalized astrocyte cell line derived from a rat (claim 5), and a temperature-sensitive mutant SV40. The immortalized astrocyte cell line derived from the transgenic rat into which the large T antigen gene of tsA58 has been introduced is TR-AST932 (FEPMBP-6283). A method for preparing a blood-brain barrier reconstruction model according to claim 5 6.) or rat immortalized brain capillary pericyte cell line derived from the, large temperature sensitive mutant SV 4 0 t SA 5 8 3. The method for preparing a blood-brain barrier remodeling model according to claim 2, which is an immortalized brain capillary pericyte cell line derived from a transgenic rat into which a T antigen gene has been introduced. An immortalized brain capillary pericyte cell line derived from a transgenic rat into which the large T antigen gene of the temperature-sensitive mutant SV40tsA58 was introduced was TR-PCT1 (FERMBP-7204). 8. The method for producing a blood-brain barrier reconstruction model according to claim 7, wherein the co-culture is performed by using an immortalized brain capillary endothelial cell line and an immortalized first mouth site cell. Culturing the cell line in a non-contact state, or in a non-contact state with an immortalized cerebral capillary endothelial cell line, an immortalized ostium-site cell line, and an immortalized cerebral capillary pericyte cell line. The blood-brain barrier according to any one of claims 1 to 8, which is a system co-culture. The present invention relates to a method for producing a construction model (claim 9).
また本発明は、 請求項 1〜 9のいずれか記載の血液脳関門再構築モデ ルを用いるスクリーニング方法であって、共培養中又は共培養の前後に、 被検物質を不死化脳毛細血管内皮細胞株と接触させ、 血液脳関門のマー カー遺伝子の発現の程度を測定 · 評価することを特徴とする血液脳関門 形成促進又は抑制物質のスク リーニング方法 （請求項 1 0 ) や、 請求項 1〜 9のいずれか記載の血液脳関門再構築モデルを用いるスクリ一ニン グ方法であって、 共培養中又は共培養の前後に、 被検物質と血液脳関門 透過物質又は血液脳関門非透過物質とを不死化脳毛細血管内皮細胞株と 接触させ、 これら血液脳関門透過物質又は血液脳関門非透過物質の不死 化脳毛細血管内皮細胞内への透過の程度を測定 · 評価することを特徴と する血液脳関門透過促進又は抑制物質のスクリーニング方法 （請求項 1 1 ) や、 請求項 1〜 9のいずれか記載の血液脳関門再構築モデルを用い るスク リーニング方法であって、 共培養中又は共培養の前後に、 被検物 質を不死化脳毛細血管内皮細胞株と接触させ、 不死化脳毛細血管内皮細
胞内への被検物質の透過の程度を測定 · 評価することを特徴とする血液 脳関門透過又は非透過物質のスク リーニング方法 （請求項 1 2 ) や、 請 求項 1 0記載の血液脳関門形成促進又は抑制物質のスク リ一二ング方法 により得られる血液脳関門形成促進物質 （請求項 1 3 ) や、 請求項 1 0 記載の血液脳関門形成促進又は抑制物質のスク リ一二ング方法により得 られる血液脳関門形成抑制物質 （請求項 1 4 ) や、 請求項 1 1記載の血 液脳関門透過促進又は抑制物質のスクリーニング方法により得られる血 液脳関門透過促進物質 （請求項 1 5 ) や、 請求項 1 1記載の血液脳関門 透過促進又は抑制物質のスクリーニング方法により得られる血液脳関門 透過抑制物質 （請求項 1 6 ) や、 請求項 1 2記載の血液脳関門透過又は 非透過物質のスク リーニング方法により得られる血液脳関門透過物質 (請求項 1 7 ) や、 請求項 1 2記載の血液脳関門透過又は非透過物質の スクリーニング方法により得られる血液脳関門非透過物質（請求項 1 8 ) に関する。 The present invention also provides a screening method using the blood-brain barrier reconstruction model according to any one of claims 1 to 9, wherein the test substance is immortalized during or before or after co-culture. A method for screening a substance that promotes or suppresses the formation of the blood-brain barrier, which is characterized by measuring and evaluating the degree of expression of a marker gene at the blood-brain barrier by contacting with a cell line (claim 10); A screening method using the blood-brain barrier reconstruction model according to any one of claims 9 to 9, wherein the test substance and the blood-brain barrier permeable substance or the blood-brain barrier impervious substance during or before and after co-culture. And contacting it with an immortalized brain capillary endothelial cell line, and measuring and evaluating the degree of permeation of these blood-brain barrier permeable substances or blood-brain barrier non-permeable substances into the immortalized brain capillary endothelial cells. The blood-brain barrier A screening method for a promoting or inhibitory substance (Claim 11) or a screening method using the blood-brain barrier reconstruction model according to any one of Claims 1 to 9, wherein the screening method is performed during or after co-culture. Contacting the test substance with the immortalized brain capillary endothelial cell line; A method for screening a blood-brain barrier permeating or non-permeating substance (Claim 12), and a blood brain according to Claim 10 characterized by measuring and evaluating the degree of penetration of a test substance into a cell. The blood-brain barrier formation promoting substance (Claim 13) obtained by the method for screening a barrier formation promoting or suppressing substance, and the screening of the blood-brain barrier formation promoting or suppressing substance according to claim 10 The blood-brain barrier formation inhibitory substance obtained by the method (Claim 14), and the blood-brain barrier permeation enhancer obtained by the method for screening for a blood-brain barrier permeation promoting or inhibiting substance according to Claim 11 (Claim 1) 5) and the blood-brain barrier permeation-inhibiting substance (claim 16) obtained by the method for screening a blood-brain barrier permeation promoting or suppressing substance according to claim 11 or the blood-brain barrier permeation or non-permeation according to claim 12. Permeable material screen Blood-brain barrier permeable substance obtained by the method (Claim 17) and the blood-brain barrier impermeable substance obtained by the method for screening a blood-brain barrier permeable or non-permeable substance according to Claim 12 (Claim 18) About.
また本発明は、 ラッ ト由来の不死化脳毛細血管内皮細胞株と、 ラッ ト 由来の不死化ァス トロサイ ト細胞株とを共培養することにより得られる ことを特徴とする血液脳関門のマーカー遺伝子の発現が増強した不死化 脳毛細血管内皮細胞株 （請求項 1 9 ) や、 ラッ ト由来の不死化脳毛細血 管内皮細胞株と、 ラッ ト由来の不死化ァス トロサイ ト細胞株と、 ラッ ト 由来の不死化脳毛細血管周皮細胞株とを共培養することにより得られる ことを特徴とする血液脳関門のマーカー遺伝子の発現が増強した不死化 脳毛細血管内皮細胞株 （請求項 2 0 ) や、 ラッ ト由来の不死化脳毛細血 管内皮細胞株が、 温度感受性変異株 S V 4 0 t s A 5 8のラージ T抗原 遺伝子を導入したトランスジェニックラッ ト由来の不死化脳毛細血管内 皮細胞株であることを特徴とする請求項 1 9又は 2 0記載の血液脳関門 のマーカー遺伝子の発現が増強した不死化脳毛細血管内皮細胞株 （請求
項 2 1 ) や、 温度感受性変異株 S V 4 0 t s A 5 8のラージ T抗原遺伝 子を導入したトランスジエニックラッ ト由来の不死化脳毛細血管内皮細 胞株が、 T R— B B B 1 3 (F E P M B P— 6 8 7 3 ) であることを 特徴とする請求項 2 1記載の血液脳関門のマーカー遺伝子の発現が増強 した不死化脳毛細血管内皮細胞株 （請求項 2 2 ) や、 ラッ ト由来の不死 化ァス ト口サイ ト細胞株が、 温度感受性変異株 S V 4 0 t s A 5 8のラ —ジ T抗原遺伝子を導入したトランスジエニックラッ ト由来の不死化ァ ス ト口サイ ト細胞株であることを特徴とする請求項 1 9又は 2 0記載の 血液脳関門のマーカー遺伝子の発現が増強した不死化脳毛細血管内皮細 胞株 （請求項 2 3 ) や、 温度感受性変異株 S V 4 0 t s A 5 8のラージ T抗原遺伝子を導入したトランスジエニックラッ ト由来の不死化ァス ト 口サイ ト細胞株が、 T R— A S T 9 3 2 (F E P M B P— 6 2 8 3 ) であることを特徴とする請求項 2 3記載の血液脳関門のマーカ一遺伝子 の発現が増強した不死化脳毛細血管内皮細胞株 （請求項 2 4 ) や、 ラッ 卜由来の不死化脳毛細血管周皮細胞株が、 温度感受性変異株 S V 4 0 t s A 5 8のラージ T抗原遺伝子を導入したトランスジエニックラッ ト由 来の不死化脳毛細血管周皮細胞株であることを特徴とする請求項 2 0記 載の血液脳関門のマーカー遺伝子の発現が増強した不死化脳毛細血管内 皮細胞株 （請求項 2 5 ) や、 温度感受性変異株 S V 4 0 t s A 5 8のラ ージ T抗原遺伝子を導入したトランスジェニックラッ ト由来の不死化脳 毛細血管周皮細胞株が、 T R— P C T 1 (F E RM B P— 7 0 2 4 ) であることを特徴とする請求項 2 5記載の血液脳関門のマーカー遺伝子 の発現が増強した不死化脳毛細血管内皮細胞株 （請求項 2 6 ) や、 共培 養が、 不死化脳毛細血管内皮細胞株と不死化ァス ト口サイ ト細胞株とを 非接触状態で、 又は不死化脳毛細血管内皮細胞株と不死化ァス ト口サイ ト細胞株及び不死化脳毛細血管周皮細胞株とを非接触状態で培養する、
非接触系共培養であることを特徴とする請求項 1 9〜 2 6のいずれか記 載の血液脳関門のマーカー遺伝子の発現が増強した不死化脳毛細血管内 皮細胞株 （請求項 2 7 ) や、 血液脳関門のマーカー遺伝子が、 アルカリ フォスファ夕一ゼ遺伝子、 ァ 一グル夕ミールトランスぺプチダ一ゼ遺伝 子、 G 1 u t 1遺伝子から選ばれる 1種又は 2種以上の遺伝子であるこ とを特徴とする請求項 1 9〜 2 7のいずれか記載の血液脳関門のマーカ 一遺伝子の発現が増強した不死化脳毛細血管内皮細胞株 （請求項 2 8 ) や、 アルカリフォスファターゼ遺伝子の発現が、 共培養することなく単 独培養したときと比べて 6倍以上増強したことを特徴とする請求項 2 8 記載の血液脳関門のマーカー遺伝子の発現が増強した不死化脳毛細血管 内皮細胞株 （請求項 2 9 ) や、 ァ ーダル夕ミールトランスべプチダーゼ 遺伝子の発現が、 共培養することなく単独培養したときと比べて 2倍以 上増強したことを特徴とする請求項 2 8又は 2 9記載の血液脳関門のマ —カー遺伝子の発現が増強した不死化脳毛細血管内皮細胞株 （請求項 3 0 ) や、 G 1 u t 1遺伝子の発現が、 共培養することなく単独培養した ときと比べて 1 0 0倍以上増強したことを特徴とする請求項 2 8〜 3 0 のいずれか記載の血液脳関門のマーカー遺伝子の発現が増強した不死化 脳毛細血管内皮細胞株 （請求項 3 1 ) に関する。 The present invention also provides a blood-brain barrier marker obtained by co-culturing a rat-derived immortalized brain capillary endothelial cell line and a rat-derived immortalized astrocyte cell line. Immortalized brain capillary endothelial cell lines with enhanced gene expression (Claim 19), rat-derived immortalized brain capillary endothelial cell lines, and rat-derived immortalized astrocyte cell lines An immortalized brain capillary endothelial cell line having enhanced expression of a blood brain barrier marker gene, which is obtained by co-culturing a rat-derived immortalized brain capillary pericyte cell line. 20) and an immortalized cerebral capillary endothelial cell line derived from a rat was transformed into an immortalized cerebral capillary derived from a transgenic rat into which the large T antigen gene of the temperature-sensitive mutant SV40tsA58 was introduced. Endothelial cell line Features to claim 1 9 or 0 Expression of the marker gene of the blood brain barrier described enhanced immortalized brain capillary endothelial cell line (according Item 21) and the immortalized cerebral capillary endothelial cell line derived from the transgenic rat into which the large T antigen gene of the temperature-sensitive mutant SV40tsA58 was introduced were TR-BBB13 ( 21. An immortalized brain capillary endothelial cell line having enhanced expression of a marker gene for the blood-brain barrier according to claim 21, characterized in that it is FEPMBP-6873). The immortalized ostium site cell line is a transgenic rat derived from the transgenic rat into which the temperature-sensitive mutant SV40tsA58 radio-T antigen gene has been introduced. 21. An immortalized brain capillary endothelial cell strain with enhanced expression of a marker gene for the blood brain barrier according to claim 19 or claim 20, wherein the temperature-sensitive mutant SV is a strain. An immortalized mouse derived from a transgenic rat into which the large T antigen gene of 40 ts A58 was introduced. The immortalized brain capillary according to claim 23, wherein the oral site cell line is TR-AST932 (FEPMBP-6282), wherein the expression of the marker gene of the blood brain barrier is enhanced. A vascular endothelial cell line (Claim 24) or a rat-derived immortalized cerebral capillary pericyte cell line is a transgenic rat into which the large T antigen gene of the temperature-sensitive mutant SV40tsA58 was introduced. 20. An immortalized brain capillary endothelial cell line having enhanced expression of a marker gene of the blood-brain barrier according to claim 20, wherein the immortalized brain capillary pericyte cell line is derived from 25) and an immortalized brain capillary pericyte cell line derived from a transgenic rat into which the large T antigen gene of the temperature-sensitive mutant SV40tsA58 was introduced was TR-PCT1 (FERM BP-7024), wherein the expression of the marker gene for the blood brain barrier according to claim 25 is increased. The immortalized cerebral capillary endothelial cell line (Claim 26) or the co-culture, the immortalized cerebral capillary endothelial cell line and the immortalized ostium site cell line in non-contact or immortalized state Culturing an immortalized cerebral capillary endothelial cell line, an immortalized ostium site cell line and an immortalized cerebral capillary pericyte cell line in a non-contact state; An immortalized brain capillary endothelial cell line having enhanced expression of a blood-brain barrier marker gene according to any one of claims 19 to 26, which is a non-contact co-culture (claim 27) ) Or the blood-brain barrier marker gene is one or more genes selected from the group consisting of alkaline phosphatase gene, alginate meal transpeptidase gene, and G1ut1 gene. An immortalized brain capillary endothelial cell line in which expression of the marker for the blood-brain barrier according to any one of claims 19 to 27 is enhanced, and expression of the alkaline phosphatase gene is enhanced. The immortalized brain capillary endothelial cell line according to claim 28, wherein the expression of a marker gene for the blood-brain barrier is enhanced by 6 times or more as compared with the case of single culture without co-culture. Claim 29) and the key 30. The marker for the blood-brain barrier according to claim 28 or 29, wherein the expression of the transgenic evening meal transpeptidase gene is enhanced by at least two times as compared with the case of single culture without co-culture. Immortalized cerebral capillary endothelial cell line with enhanced gene expression (Claim 30) and G1ut1 gene expression was enhanced 100-fold or more compared to single culture without co-culture An immortalized brain endothelial cell line having enhanced expression of a blood-brain barrier marker gene according to any one of claims 28 to 30 (claim 31).
また本発明は、 被検物質と、 請求項 1 9〜 3 1 のいずれか記載の血液 脳関門のマーカー遺伝子の発現が増強した不死化脳毛細血管内皮細胞株 とを接触させ、 血液脳関門のマーカ一遺伝子の発現増強の程度を測定 · 評価することを特徴とする血液脳関門形成促進又は抑制物質のスクリ一 ニング方法 （請求項 3 2 ) や、 被検物質と血液脳関門透過物質又は血液 脳関門非透過物質と、 請求項 1 9 〜 3 1 のいずれか記載の血液脳関門の マーカー遺伝子の発現が増強した不死化脳毛細血管内皮細胞株とを接触 させ、 これら血液脳関門透過物質又は血液脳関門非透過物質の該不死化
脳毛細血管内皮細胞内への透過の程度を測定 · 評価することを特徴とす る血液脳関門透過促進又は抑制物質のスク リーニング方法（請求項 3 3 ) や、 被検物質と、 請求項 1 9〜 3 1 のいずれか記載の血液脳関門のマ一 カー遺伝子の発現が増強した不死化脳毛細血管内皮細胞株とを接触させ. 該不死化脳毛細血管内皮細胞内への被検物質の透過の程度を測定 · 評価 することを特徴とする血液脳関門透過又は非透過物質のスク リーニング 方法 （請求項 3 4 ) や、 請求項 3 2記載の血液脳関門形成促進又は抑制 物質のスク リーニング方法により得られる血液脳関門形成促進物質 （請 求項 3 5 ) や、 請求項 3 2記載の血液脳関門形成促進又は抑制物質のス クリ一ニング方法により得られる血液脳関門形成抑制物質（請求項 3 6 ) や、 請求項 3 3記載の血液脳関門透過促進又は抑制物質のスクリーニン グ方法により得られる血液脳関門透過促進物質 （請求項 3 7 ) や、 請求 項 3 3記載の血液脳関門透過促進又は抑制物質のスクリーニング方法に より得られる血液脳関門透過抑制物質 （請求項 3 8 ) や、 請求項 3 4記 載の血液脳関門透過又は非透過物質のスク リ一二ング方法により得られ る血液脳関門透過物質 （請求項 3 9 ) や、 請求項 3 4記載の血液脳関門 透過又は非透過物質のスク リーニング方法により得られる血液脳関門非 透過物質 （請求項 4 0 ) に関する。 図面の簡単な説明 The present invention also provides a test substance and an immortalized brain capillary endothelial cell line in which the expression of a marker gene for the blood brain barrier according to any one of claims 19 to 31 is contacted, whereby the blood brain barrier is A screening method for a substance promoting or inhibiting the formation of the blood-brain barrier, which comprises measuring and evaluating the degree of expression of the marker gene (Claim 32); and a test substance and a substance permeating the blood-brain barrier or blood. Contacting a brain barrier impermeable substance with an immortalized brain capillary endothelial cell line having enhanced expression of a blood brain barrier marker gene according to any one of claims 19 to 31; The immortalization of blood-brain barrier impermeable material A method for screening a substance promoting or inhibiting blood-brain barrier permeation, which comprises measuring and evaluating the degree of permeation into brain capillary endothelial cells (Claim 33); a test substance; 9) contacting the immortalized brain capillary endothelial cell line with enhanced expression of the blood-brain barrier marker gene according to any one of 9 to 31. A method for screening a blood-brain barrier permeating or non-permeating substance, characterized by measuring and evaluating the degree of permeation (Claim 34), and a screening method for a substance promoting or suppressing blood-brain barrier formation according to Claim 32. A blood-brain barrier formation-promoting substance obtained by the method (claim 35) or a blood-brain barrier formation-suppressing substance obtained by the method for screening a blood-brain barrier formation-promoting or suppressing substance according to claim 32 (claim). Item 36) and claim 33 The blood-brain barrier penetration-enhancing substance (Claim 37) obtained by the screening method of the blood-brain barrier permeation-promoting or inhibitory substance, or the blood-brain barrier penetration-enhancing or inhibiting substance screening method of Claim 33. A blood-brain barrier permeation inhibitor (Claim 38) or a blood-brain barrier permeant obtained by the method for screening a blood-brain barrier permeable or non-permeable material according to Claim 34 (Claim 3). 9) and a blood-brain barrier impermeable material (claim 40) obtained by the method for screening a blood-brain barrier permeated or non-permeable material according to claim 34. BRIEF DESCRIPTION OF THE FIGURES
第 1図は、 本発明における非接触系共培養法の概略を示す図である。 第 2図は、 本発明の共培養による血液脳関門再構築モデルの A L P活 性の結果を示す図である。 FIG. 1 is a diagram schematically showing a non-contact type co-culture method in the present invention. FIG. 2 is a view showing the results of ALP activity of a blood-brain barrier reconstruction model by co-culture according to the present invention.
第 3図は、 本発明の共培養による血液脳関門再構築モデルのァ G T P活性の結果を示す図である。 FIG. 3 shows the results of α GTP activity of the blood-brain barrier remodeling model by co-culture of the present invention.
第 4図は、 本発明の共培養による血液脳関門再構築モデルにおける G
L U T - 1発現結果を示す図である。 発明を実施するための最良の形態 FIG. 4 shows G in the blood-brain barrier reconstruction model by co-culture of the present invention. FIG. 9 is a view showing a result of LUT-1 expression. BEST MODE FOR CARRYING OUT THE INVENTION
本発明の血液脳関門再構築モデルの作製方法は、 ラッ ト由来の不死化 脳毛細血管内皮細胞株と、 ラッ ト由来の不死化ァス トロサイ ト細胞株と を共培養することや、 ラッ ト由来の不死化脳毛細血管内皮細胞株と、 ラ ッ ト由来の不死化ァス トロサイ ト細胞株と、 ラッ ト由来の不死化脳毛細 血管周皮細胞株とを共培養することを特徴とする。 また、 本発明の血液 脳関門のマーカー遺伝子の発現が増強した不死化脳毛細血管内皮細胞株 は、 ラッ ト由来の不死化脳毛細血管内皮細胞株と、 ラッ ト由来の不死化 ァス トロサイ ト細胞株とを共培養することにより得られることや、 ラッ 卜由来の不死化脳毛細血管内皮細胞株と、 ラッ 卜由来の不死化ァス ト口 サイ 卜細胞株と、 ラッ ト由来の不死化脳毛細血管周皮細胞株とを共培養 することにより得られることを特徴とする。 The method for preparing the blood-brain barrier remodeling model of the present invention includes co-culturing a rat-derived immortalized brain capillary endothelial cell line with a rat-derived immortalized astrocyte cell line, Co-cultured with an immortalized brain capillary endothelial cell line derived from a rat, an immortalized astrocyte cell line derived from a rat, and an immortalized brain capillary pericyte cell line derived from a rat. . In addition, the immortalized brain capillary endothelial cell line having enhanced expression of the blood brain barrier marker gene of the present invention includes a rat-derived immortalized brain capillary endothelial cell line and a rat-derived immortalized astrocyte. What can be obtained by co-culturing with a cell line, rat-derived immortalized cerebral capillary endothelial cell line, rat-derived immortalized first mouth cell line, rat-derived immortalization It is obtained by co-culturing with a brain capillary pericyte line.
上記ラッ ト由来の不死化脳毛細血管内皮細胞株、 不死化ァス ト口サイ ト細胞株、 及び不死化脳毛細血管周皮細胞株としては、 特に制限される ものではないが、 それぞれ正常な脳毛細血管内皮細胞、 ァス トロサイ ト 細胞及び脳毛細血管周皮細胞が本来有する機能や性状を保持したまま不 死化細胞として樹立された細胞株が好ましい。 これら不死化細胞株の樹 立方法としては特に制限されないが、 例えば、 S V 4 0の温度感受性突 然変異株 t s A 5 8のラージ T抗原遺伝子を導入した トランスジェニッ クラッ 卜から得られる不死化脳毛細血管内皮細胞株、 不死化ァス トロサ ィ ト細胞株及び不死化脳毛細血管周皮細胞株が、 温度条件によりその増 殖を制御しうる、すなわち 3 3〜 3 7 °Cにおいて永久的増殖能を保持し、 3 9 °Cにおいては増殖を停止するため、 細胞固有の分化形質の発現を制 御することができることから好ましい。
かかる S V 4 0の温度感受性突然変異株 t s A 5 8のラージ T抗原遺 伝子を導入した トランスジエニックラッ ト由来の不死化脳毛細血管内皮 細胞株としては、 血液脳関門の酵素マ一力一であるアル力リ フォスファ 夕ーゼ （A L P) ゃァーダル夕ミールトランスぺプチダーゼ （ァ GT Ρ)、へキソース輸送担体である G L UT - 1等を発現する細胞株 T R— B B B 1、 TR— B B B 5、 T R— B B B 6、 TR— B B B 1 1、 TR - Β Β Β 1 3等を具体的に例示することができる。 上記細胞株 T R— Β Β Β 1 3は、 ブ夕ぺス ト条約に基づいて経済産業省産業技術総合研究所 生命工学工業技術研究所 （日本国茨城県つくば巿東一丁目 1番 3号 郵 便番号 3 0 5— 8 5 6 6 ) に受託番号 N I B H F E RM B P— 6 8 7 3として寄託されている。 The immortalized cerebral capillary endothelial cell line, the immortalized ostium site cell line, and the immortalized cerebral capillary pericyte cell line derived from the above-mentioned rat are not particularly limited, but are each normal. A cell line established as an immortalized cell while retaining the functions and properties inherent in brain capillary endothelial cells, astrosite cells, and brain capillary pericytes is preferable. The method of establishing these immortalized cell lines is not particularly limited.For example, immortalized cells obtained from transgenic rats into which the large T antigen gene of the temperature-sensitive mutant tsA58 of SV40 has been introduced. Brain capillary endothelial cell line, immortalized astrocyte cell line and immortalized brain capillary pericyte cell line can control their growth by temperature conditions, i.e. permanent at 33-37 ° C It is preferable because it retains the proliferative ability and stops the growth at 39 ° C., so that the expression of differentiation traits specific to cells can be controlled. The immortalized brain capillary endothelial cell line derived from the transgenic rat into which the large T antigen gene of the SV40 temperature-sensitive mutant tsA58 has been introduced includes the blood-brain barrier enzyme. Cell lines TR-BBB1 and TR-BBB that express the lipophilic enzyme phospholipase (ALP) ゃ adal mirmir transpeptidase (ァ GT GT) and the hexose transporter GLUT-1 5, TR—BBB 6, TR—BBB 11 and TR—Β Β 3 13 can be specifically exemplified. The above cell line TR— TR Β 3 3 13 is based on the Busyeast Treaty, the Institute of Biotechnology, Industrial Technology Research Institute, Ministry of Economy, Trade and Industry (1-3 1-3 Tsukuba East, Ibaraki, Japan) Deposit No. NIBHFE RM BP—6 873 3 on flight number 3 05—8 5 6 6).
また、 S V 4 0の温度感受性突然変異株 t s A 5 8のラージ T抗原遺 伝子を導入したトランスジエニックラッ ト由来の不死化ァス トロサイ ト 細胞株としては、 N a +依存性 L—グルタミン酸トランスポー夕一等の 発現能を有する細胞株 T R— A S T 3 2、 T R— A S T 8 1 1、 TR— A S T 9 1 2、 TR— A S T 9 3 2、 T R— A S T 9 4 3等を具体的に 例示することができる。 上記細胞株 T R— A S T 9 3 2は、 ブタペス ト 条約に基づいて経済産業省産業技術総合研究所生命工学工業技術研究所 (曰本国茨城県つく ば巿東一丁目 1番 3号 郵便番号 3 0 5 - 8 5 6 6 ) に受託番号 N I B H F E RM B P— 6 2 8 3 として寄託されて いる。 同様に、 S V 4 0の温度感受性突然変異株 t s A 5 8のラージ T 抗原遺伝子を導入した トランスジエニックラッ ト由来の不死化脳毛細血 管周皮細胞株としては、 P D G F受容体 3及びアンギオポェチン 1等 の発現能を有する細胞株 T R— P C T 1、 T R— P C T 2等を具体的に 例示することができる。 上記細胞株 T R— P C T 1は、 ブタペス ト条約 に基づいて経済産業省産業技術総合研究所生命工学工業技術研究所 （日 In addition, as an immortalized astrocyte cell line derived from a transgenic rat into which a large T antigen gene of the SV40 temperature-sensitive mutant tsA58 was introduced, Na + -dependent L- Specific cell lines capable of expressing glutamate transport such as TR-AST32, TR-AST811, TR-AST912, TR-AST932, TR-AST944, etc. Can be exemplified. Based on the Budapest Treaty, the above-mentioned cell line TR—AST 932 was obtained from the Ministry of Economy, Trade and Industry, National Institute of Advanced Industrial Science and Technology, Institute of Biotechnology and Industrial Technology (1-3, Tsukuba, Higashi, Ibaraki, Japan). 5-8 5 6 6) and deposited under the accession number NIBHFE RM BP—6283. Similarly, the immortalized cerebral capillary pericyte cell line derived from the transgenic rat into which the large T antigen gene of the temperature-sensitive mutant tsA58 of SV40 has been introduced includes PDGF receptor 3 and angiopoietin. Specific examples include cell lines TR-PCT1, TR-PCT2, etc., which have an expression ability of 1. Based on the Budapest Treaty, the above cell line TR—PCT1 was developed by the Ministry of Economy, Trade and Industry,
12 訂正された用紙 （規則 91)
本国茨城県つくば巿東一丁目 1番 3号 郵便番号 3 0 5— 8 5 6 6 ) に 受託番号 N I B H F E RM B P— 7 0 2 4として寄託されている。 本発明における血液脳関門再構築モデルは、 ラッ ト由来の不死化脳毛 12 Corrected Form (Rule 91) It has been deposited with the postal code 305-5-8566-6), Tsukuba, Higashi 1-3-chome, Ibaraki, Japan, under the accession number NIBHFE RM BP-7024. The blood-brain barrier reconstruction model in the present invention is a rat-derived immortalized brain hair.
12/1 12/1
訂正された用紙 (細 IJ91)
細血管内皮細胞株と、 ラッ ト由来の不死化ァス トロサイ ト細胞株とを共 培養することや、 ラッ ト由来の不死化脳毛細血管内皮細胞株と、 ラッ ト 由来の不死化ァス トロサイ ト細胞株と、 ラッ ト由来の不死化脳毛細血管 周皮細胞株とを共培養することによって作製することができる。 共培養 は、 これら細胞株同士を接触状態で培養することにより、 又は、 不死化 脳毛細血管内皮細胞株と不死化ァス トロサイ ト細胞株、 若しくは不死化 脳毛細血管内皮細胞株と不死化ァス トロサイ ト細胞株及び不死化脳毛細 血管周皮細胞株とを、 例えば液性因子などは透過することができるが細 胞は透過することができない膜を介して、 非接触状態で培養することに より行う ことができる。 このような膜を介する非接触状態での共培養に より、 不死化脳毛細血管内皮細胞株だけを他の細胞から簡単に分離する ことができるばかりでなく、 種々の孔サイズの膜を用いることにより、 細胞相互間の情報伝達を担う液性因子の分子量を推定することができる, また、 本発明の血液脳関門のマーカ一遺伝子、 例えばアルカリフォス ファタ一ゼ遺伝子、 ァ—ダルタミールトランスぺプチダ一ゼ遺伝子、 G 1 u t 1遺伝子の発現が増強した不死化脳毛細血管内皮細胞株は、 前記 S V 4 0の温度感受性突然変異株 t s A 5 8のラージ T抗原遺伝子を導 入したトランスジェニックラッ ト由来の不死化脳毛細血管内皮細胞株を. 前記不死化ァス トロサイ ト細胞株、 又は該不死化ァス トロサイ ト細胞株 と前記不死化脳毛細血管周皮細胞株と共培養することにより得ることが できる。 かかる血液脳関門のマーカ一遺伝子の発現が増強した不死化脳 毛細血管内皮細胞株としては、 共培養後も、 共培養することなく単独培 養したときと比べて、 アルカリフォスファ夕ーゼ遺伝子の発現が 6倍以 上、 ァーダル夕ミールトランスべプチダーゼ遺伝子の発現が 2倍以上、 G 1 u t 1遺伝子の発現が 1 0 0倍以上増強された性状を維持した不死 化脳毛細血管内皮細胞株が好ましい。 さらに、 多孔性平面膜上で単層培 Corrected form (Fine IJ91) Co-culturing a microvascular endothelial cell line with a rat-derived immortalized astrocyte cell line, or using a rat-derived immortalized brain capillary endothelial cell line and a rat-derived immortalized astrocyte cell line And a rat cell-derived immortalized brain capillary pericyte cell line. Co-culture is performed by culturing these cell lines in contact with each other, or by immortalizing cerebral capillary endothelial cell lines and immortalized astrocyte cell lines, or immortalizing cerebral capillary endothelial cell lines and immortalized cells. Non-contact culture of strocite cell line and immortalized cerebral capillary pericyte cell line through a membrane that can penetrate, for example, humoral factors, but not cells This can be done by: By co-culturing in a non-contact state through such a membrane, not only the immortalized cerebral capillary endothelial cell line can be easily separated from other cells, but also a membrane having various pore sizes can be used. Thus, the molecular weight of a humoral factor responsible for signal transmission between cells can be estimated. In addition, a marker gene of the blood-brain barrier of the present invention, for example, an alkaline phosphatase gene, adartamyl transpeptida The immortalized cerebral capillary endothelial cell line with enhanced expression of the G1ut1 gene is a transgenic rat into which the large T antigen gene of the temperature-sensitive mutant tsA58 of SV40 has been introduced. Immortalized brain capillary endothelial cell line derived from the same. Co-culture of the immortalized astrocyte cell line, or the immortalized astrocyte cell line and the immortalized brain capillary pericyte cell line. Can be obtained by: Immortalized brain capillary endothelial cell lines with enhanced expression of the marker gene of the blood-brain barrier include the alkaline phosphatase gene after co-culture compared to when cultured alone without co-culture. Immortalized cerebral capillary endothelial cell line that maintains the properties of expression of Adar evening meal transpeptidase gene more than 2 times and Gut1 gene expression more than 100 times Is preferred. In addition, a monolayer culture on a porous planar membrane
1 3
養すると細胞が相互に結合して、 表裏極性をもつ血液脳関門を試験内で 再構築することができる不死化脳毛細血管内皮細胞株が特に好ましい。 本発明における血液脳関門再構築モデルや本発明の血液脳関門のマー カー遺伝子の発現が増強した不死化脳毛細血管内皮細胞は、 血液から脳 組織への物質移行を制限している血液脳関門の研究、 すなわち、 脳の栄 養代謝研究や脳内への薬物透過研究及び血液脳関門における防御機構の 研究に活用することができる。 従って、 医薬品の安全性や有効性に関す るスクリーニング、 脳の栄養代謝及び恒常性機能障害に関連する疾患の 診断やその治療方法の開発の細胞レベルでの研究に有利である。 以下、 本発明における血液脳関門再構築モデルや本発明の血液脳関門のマーカ 一遺伝子の発現が増強した不死化脳毛細血管内皮細胞を用いた、 血液脳 関門形成促進又は抑制物質や、 血液脳関門透過促進又は抑制物質や、 血 液脳関門透過又は非透過物質のスクリーニング方法について説明する。 血液脳関門再構築モデルを用いる血液脳関門形成促進又は抑制物質の スクリーニングは、 共培養中又は共培養の前後に、 被検物質を不死化脳 毛細血管内皮細胞株と接触させ、 血液脳関門のマーカー遺伝子の発現の 程度を測定し、 被検物質が非存在の対照の場合と比較 · 評価することに より行うことができる。 また、 血液脳関門形成促進又は抑制物質のスク リーニングは、 被検物質と血液脳関門マーカー遺伝子の発現が増強した 不死化脳毛細血管内皮細胞株とを接触させ、 血液脳関門のマーカ一遺伝 子の発現増強の程度を測定し、 被検物質が非存在の対照の場合と比較 - 評価することによつても行う ことができる。 これらスクリーニングによ り得られる血液脳関門形成促進物質は、 血液脳関門形成不全に起因する 治療剤として期待することができ、 また、 これら血液脳関門形成促進又 は抑制物質は、 細胞レベルでの血液脳関門形成の研究に有用である。 血液脳関門再構築モデルを用いる血液脳関門透過促進又は抑制物質の 13 Particularly preferred are immortalized brain capillary endothelial cell lines, which, upon culture, allow the cells to bind to one another and reconstitute the blood-brain barrier with front and back polarity in the test. The blood-brain barrier remodeling model of the present invention and the immortalized brain capillary endothelial cells with enhanced expression of the blood-brain barrier marker gene of the present invention provide a blood-brain barrier that restricts the transfer of substances from blood to brain tissue. It can be used for research on nutrients and metabolism in the brain, drug permeation into the brain, and defense mechanisms at the blood-brain barrier. Therefore, it is advantageous for screening at the cellular level for screening the safety and efficacy of pharmaceuticals, diagnosing diseases associated with dysfunction of brain nutritional metabolism and homeostasis, and developing therapeutic methods therefor. Hereinafter, a blood-brain barrier remodeling model of the present invention and a blood-brain barrier formation promoting or inhibiting substance, and a blood brain using an immortalized brain capillary endothelial cell with enhanced expression of one gene of the blood-brain barrier marker of the present invention, A method for screening a substance that promotes or inhibits barrier permeation and a substance that permeates or impregnates the blood-brain barrier is described. Screening for a substance that promotes or inhibits blood-brain barrier formation using a blood-brain barrier reconstruction model involves contacting a test substance with an immortalized brain capillary endothelial cell line during or before or after co-culture, and This can be done by measuring the degree of expression of the marker gene and comparing and evaluating it with a control in which the test substance is absent. Screening of a substance that promotes or suppresses blood-brain barrier formation involves contacting a test substance with an immortalized brain capillary endothelial cell line with enhanced expression of the blood-brain barrier marker gene, and a marker for the blood-brain barrier marker. It can also be performed by measuring the degree of expression enhancement and comparing and evaluating the control in the absence of the test substance. The blood-brain barrier formation-promoting substance obtained by these screenings can be expected as a therapeutic agent due to blood-brain barrier dysfunction, and these blood-brain barrier formation-promoting or suppressing substances are at the cellular level. Useful for studying blood-brain barrier formation. Of blood-brain barrier penetration enhancer or inhibitor using blood-brain barrier reconstruction model
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スクリーニングは、 共培養中又は共培養の前後に、 既知の血液脳関門透 過物質又は血液脳関門非透過物質と被検物質とを、 不死化脳毛細血管内 皮細胞株と接触させ、 これら血液脳関門透過物質又は血液脳関門非透過 物質の不死化脳毛細血管内皮細胞内への透過の程度を測定し、 被検物質 が非存在の対照の場合と比較'評価することによって行う ことができる。 また、 血液脳関門透過促進又は抑制物質のスクリーニングは、 既知の血 液脳関門透過物質又は血液脳関門非透過物質と被検物質とを、 血液脳関 門マーカー遺伝子の発現が増強した不死化脳毛細血管内皮細胞株と接触 させ、 これら血液脳関門透過物質又は血液脳関門非透過物質の不死化脳 毛細血管内皮細胞内への透過量の程度を測定し、 被検物質が非存在の対 照の場合と比較 · 評価することによつても行うことができる。 これらス クリーニングにより得られる血液脳関門透過促進又は抑制物質は、 脳の 栄養代謝研究や脳内への薬物透過研究や血液脳関門における防御機構の 研究に活用することができ、 特に血液脳関門透過促進物質は、 中枢作用 型薬物 （抗痴呆薬、 脳腫瘍治療薬、 ウィルス治療薬、 精神神経作用薬） との併用剤として有用である。 14 Screening involves contacting a known blood-brain barrier transmissive substance or a blood-brain barrier impervious substance with a test substance with an immortalized cerebral capillary endothelial cell line during or before or after co-culture, and It can be performed by measuring the degree of penetration of the brain barrier permeating substance or the blood-brain barrier non-permeating substance into the immortalized brain capillary endothelial cells, and comparing and evaluating with the control in the absence of the test substance. . Screening for a substance that promotes or inhibits blood-brain barrier penetration involves testing a known substance that is permeable to the blood-brain barrier or a substance that is impervious to the blood-brain barrier, and a test substance, by using Contact with a capillary endothelial cell line to immortalize these blood-brain barrier permeating substances or blood-brain barrier impervious substances.Measure the extent of permeation into brain capillary endothelial cells, and check for the absence of the test substance. It can also be done by comparing and evaluating with the case of. The substances that enhance or inhibit the blood-brain barrier permeation obtained by these screens can be used for studies on nutritional metabolism in the brain, drug permeation into the brain, and research on defense mechanisms at the blood-brain barrier. The facilitator is useful as a concomitant drug with centrally acting drugs (anti-dementia drugs, drugs for treating brain tumors, drugs for viruses, and drugs for psychiatric nerves).
血液脳関門再構築モデルを用いる血液脳関門透過又は非透過物質のス クリーニングは、 共培養中又は共培養の前後に、 被検物質を不死化脳毛 細血管内皮細胞株と接触させ、 不死化脳毛細血管内皮細胞内への被検物 質の透過の程度を測定し、 被検物質が非存在の対照の場合と比較 · 評価 することによって行うことができる。 また、 血液脳関門透過又は非透過 物質のスクリーニングは、 血液脳関門マ一カー遺伝子の発現が増強した 不死化脳毛細血管内皮細胞株と被検物質とを接触させ、 該不死化脳毛細 血管内皮細胞内への被検物質の透過量の程度を測定し、 被検物質が非存 在の対照の場合と比較 · 評価することによつても行う ことができる。 こ れらスクリーニングにより得られる血液脳関門透過物質は、 前記中枢作 Screening of the blood-brain barrier permeating or non-permeating substance using the blood-brain barrier remodeling model immortalizes the test substance by contacting it with the brain capillary endothelial cell line during or before or after co-culture. The measurement can be performed by measuring the degree of penetration of the test substance into the brain capillary endothelial cells, and comparing and evaluating the control with the control without the test substance. In addition, the screening of the blood-brain barrier permeating or non-permeating substance is performed by contacting a test substance with an immortalized brain capillary endothelial cell line in which expression of the blood-brain barrier marker gene is enhanced. It can also be performed by measuring the degree of penetration of the test substance into the cells, and comparing and evaluating this with a control in which the test substance is absent. The blood-brain barrier permeable substance obtained by these screenings is
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用型薬物として期待することができ、 血液脳関門非透過物質は、 中枢で の副作用の問題がない薬物として期待することができる。 1 5 It can be expected as a drug for use in medicine, and the blood-brain barrier impermeant can be expected as a drug that does not have the problem of central side effects.
以下の実施例をもって本発明をより詳細に説明するが、 本発明はこれ ら実施例により何ら限定されるものではない。 The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.
実施例 1 (トランスジエニックラッ トの作出） Example 1 (Production of transgenic rat)
S V 4 0の温度感受性突然変異株 t s A 5 8の D NAを導入したトラ ンスジェニックラッ トは、 下記の手順で作出した。 Transgenic rats into which the DNA of the temperature sensitive mutant tsA58 of SV40 was introduced were prepared by the following procedure.
1 一 1 (導入遺伝子の調製） 1 1 1 (Preparation of transgene)
マイクロインジェクショ ンには S V 4 0の温度感受性突然変異株 t s A 5 8のゲノム D N Aを使用した。 t s A 5 8のゲノム D N Aを制限酵 素 B amH I で開環し、 p B R 3 2 2の B a m H I部位に導入し、 S ί For microinjection, genomic DNA of SV40 temperature-sensitive mutant tsA58 was used. The genomic DNA of tsA58 was opened with the restriction enzyme BamHI, introduced into the BamHI site of pBR322, and
1 I配列を S a c Π に変換して S V 4 0の複製起点 （ o r i ) を欠失す る o r i (—） とした D NAクローン p S V t s A 5 8 o r i (—） 一The DNA clone pSVtsA58ori (—) was converted from the I sequence to SacΠ and deleted as an ori (—), which deletes the SV40 origin of replication (ori).
2 (Ohno T. et al., Cytotechnology, 165-172, 1991 ； 図 1参照、） 力 ら常 法に従い導入用 DN Aを調製した。 すなわち、 大腸菌内で大量に増幅さ せることにより得られたプラスミ ド D N Aの p S V t s A 5 8 o r i (一） — 2 を制限酵素 B amH I (宝酒造社製） で消化した後、 ァガロ ース電気泳動法 （ 1 %ゲル ； ベーリ ンガー社製） により分離し、 ゲルを 溶解した後、 フエノール · クロ口ホルム処理及びエタノール沈殿処理を 行い D N Aを回収した。 回収した精製 D N Aを T Eバッファー （ 1 m M の E D TAを含む 1 O mMの T r i s — H C 1 ； p H 7. 6 ) に溶解し て 1 7 0 g /m 1 の精製 D NAを含む溶液を得た。 この D NA溶液を 注入用バッファー （ 0. I mMの E D TAを含む 1 0 mMの T r i s - H C 1 ； p H 7. 6 ) で 5 g Zm l となるように希釈して注入用 D N A溶液を調製した。 なお、 調製した DN A溶液は注入操作まで一 2 0 °C で保存した。 2 (Ohno T. et al., Cytotechnology, 165-172, 1991; see FIG. 1). DNA for introduction was prepared according to a conventional method. That is, pSVtsA58ori (I) —2 of plasmid DNA obtained by amplifying a large amount in Escherichia coli was digested with restriction enzyme BamHI (Takara Shuzo), and then agarose was digested. The DNA was separated by electrophoresis (1% gel; manufactured by Boehringer), and the gel was dissolved. The DNA was recovered by phenol-cloth form treatment and ethanol precipitation treatment. The recovered purified DNA is dissolved in TE buffer (1 OmM Tris-HC1 containing 1 mM EDTA; pH 7.6) and a solution containing 170 g / m1 purified DNA I got This DNA solution was diluted with an injection buffer (10 mM Tris-HC1 containing 0.1 mM EDTA; pH 7.6) to 5 g Zml, and the DNA solution for injection was diluted. Was prepared. The prepared DNA solution was stored at 120 ° C until injection.
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1 - 2 ( トランスジエニックラッ 卜の作出） 1 6 1-2 (Transgenic Rat Creation)
ラッ ト前核期受精卵への上記調製した注入用 D N A溶液のマイクロイ ンジェクショ ンは下記の要領で行った。 性成熟した 8週齢のゥイス夕一 ラッ トを明暗サイクル 1 2時間 （ 4 ： 0 0〜 1 6 ： 0 0を明時間）、 温度 2 3 ± 2 °C、 湿度 5 5 ± 5 %で飼育し、 膣スメァにより雌の性周期を観 察して、 ホルモン処理日を選択した。 まず、 雌ラッ トにより 1 5 0 I U / k gの妊馬血清性性腺刺激ホルモン （日本ゼンャク社製 ； PM S全薬 Microinjection of the above prepared DNA solution for injection into fertilized eggs at the pronuclear stage was performed as follows. Sexually mature 8-week-old chairs are bred in a light-dark cycle for 12 hours (4: 00 to 16: 00 for light hours), at a temperature of 23 ± 2 ° C, and a humidity of 55 ± 5%. Then, the female estrous cycle was observed by a vaginal smear, and the date of hormone treatment was selected. First, 150 IU / kg of pregnant female serum gonadotropin (manufactured by Nippon Zenyaku; PMS
(pregnant mare serum gonadotropin： P M )) ¾ S复 S空内投与し、 その 4 8時間後に 7 5 I UZ k gのヒ ト絨毛性性腺刺激ホルモン （三共 8蔵 ¾社製 ； ノ。へ口一ク ノ (human chorionic gonadotropin： h C Gノ ) を投与して過剰排卵処理を行った後、 雄との同居により交配を行った. h C G投与 3 2時間後に卵管灌流により前核期受精卵を採取した。 卵管 灌流及び卵の培養には mK R B液 （Toyoda Y. and Chang M.C., J. Reprod. Fertil., 36, 9-22, 1974) を使用した。 採取した受精卵を 0. 1 % のヒアルロニダーゼ （シグマ社製 ； Hyaluronidase Typel-S) を含む m KR B液中で 3 7 °C、 5分間の酵素処理を行い卵丘細胞を除去した後、 mKR B液で 3回洗浄して酵素を除去し、 D N A注入操作まで C 02— インキュベーター内 （ 5 %の C 02— 9 5 %の A i r， 3 7 °C、 飽和湿 度） に保存した。 この様にして準備したラッ ト受精卵の雄性前核に前記 D NA溶液を注入した。 注入した 2 2 8個の卵を 9匹の仮親に移植して 出産させ 8 0匹の産仔を得た。 注入 D N Aのラッ トへの導入は、 離乳直 後に断尾して得た尾より調製した D N Aを P C R法により検定した [使 用プライマ一 ； t s A 5 8— 1 A， 5 ' - T C C TAATG TG C AG T CAGGT G- 3 ' ( 1 3 6 5〜 1 3 8 4部位に相当：配列番号 1 )、 t s A 5 8 - 1 B , 5 ' AT GA C GAG C TTT GG C AC TT G - 3 ' ( 1 5 7 1〜 1 5 9 0部位に相当 ： 配列番号 2 )]。 その結果、 遺 (pregnant mare serum gonadotropin: PM)) ¾S 复 S was administered in the air, and 48 hours later, 75 IUZ kg of human chorionic gonadotropin (manufactured by Sankyo 8Kura Co., Ltd .; no.) After superovulation treatment with human chorionic gonadotropin (hCGno), the mice were mated by cohabitation with males. 3 hours after hCG administration, fertilized eggs at pronuclear stage were collected by fallopian tube perfusion Oviduct Perfusion and egg culture were performed using mK RB solution (Toyoda Y. and Chang MC, J. Reprod. Fertil., 36, 9-22, 1974). After removing cumulus cells by performing enzyme treatment at 37 ° C for 5 minutes in mKR B solution containing Hyaluronidase (Sigma; Hyaluronidase Typel-S), the enzyme was washed three times with mKR B solution. was removed, C 0 2 to DNA injection operation - an incubator. - was stored in (5% C 0 2 9 5% a ir, 3 7 ° C, saturated humidity) rats were prepared in this way Receiving The above-mentioned DNA solution was injected into the male pronucleus of the eggs The injected 228 eggs were transplanted into 9 foster mothers to give birth to obtain 80 offspring. For the introduction, DNA prepared from the tail obtained by cutting immediately after weaning was assayed by PCR. [Primer used; tsA58-1A, 5'-TCC TAATG TG C AG T CAGGT G-3 '(Corresponding to 1365 to 13844 sites: SEQ ID NO: 1), tsA58-1B, 5' ATGA C GAG C TTT GGC AC TTT G-3 '(1570- Equivalent to 159 sites: SEQ ID NO: 2)].
1 7
伝子導入の認められた 2 0匹 （雄 6匹、 雌 8匹、 生別不明 6匹） の産仔 の中から性成熟期間を経過する 1 2週齢まで生存した 1 1 ラインの トラ ンスジエニックラッ ト （雄ライン ： # 0 7 — 2， # 0 7 — 5， # 0 9 — 6 , # 1 2 — 3， # 1 9 — 5， 雌ライン ： # 0 9 _ 7， # 1 1 — 6， # 1 2 - 5 , # 1 2 — 7， # 1 8 — 5， # 1 9 — 8 ) を得た。 これらの G 。世代の トランスジエニックラッ トとゥイス夕一ラッ トを交配し、 雄フ アウンダ一の 2 ライン （# 0 7 — 2 , # 0 7 — 5 ) と雌フアウンダ一の 3 ライン （ # 0 9— 7， # 1 1 — 6， # 1 9 — 8 ) において次世代以降 への遺伝子の伝達を確認した。 1 7 Of the 20 offspring (6 males, 8 females, and 6 unidentified pups) that had been transduced, 11 lines survived until the age of 2 weeks after sexual maturity. Genetic rat (male line: # 07-7, # 07-7, 5, # 09-2, 3, # 12-3, # 19-9, 5, female line: # 09_7, # 11) — 6, # 1 2-5, # 1 2 — 7, # 1 8 — 5, # 1 9 — 8). These G. The transgenic rat of the generation is crossed with the Chinese rat, and two lines (# 07--2, # 07--5) of male female and three lines (# 09--7) of female female. , # 11-6 and # 19-8) confirmed the gene transfer to the next generation and beyond.
実施例 2 (脳毛細血管内皮細胞の調製） Example 2 (Preparation of brain capillary endothelial cells)
2 - 1 (脳毛細血管内皮細胞の分離） 2-1 (isolation of brain capillary endothelial cells)
実施例 1で得られた S V 4 0の温度感受性突然変異株 t s A 5 8のラ ージ T抗原遺伝子を導入したトランスジエニックラッ ト （ 1 匹） より大 脳を摘出した。 クリーンベンチ内で摘出した大脳を氷冷した調整用緩衝 液 （ 1 0 m Mの H e p e s 、 l O O U Z m l の benzylpenicillin potassium, 1 0 0 /i g z m l の streptomycin sulfate , 0. 5 %のゥ シ血清アルブミンを含む H B S S ) でよく洗浄した後、 大脳を l〜 2 m m 3 に細切 し、 l m 1 用テーバー型テ フ ロ ン製ホモゲナイ ザー (WHEATON社製） に移し、 1 m 1 の氷冷した調整用緩衝液を加え、 4 回のアップダウンのス トロークを行い組織をホモゲナイズしてスラリー を得た。 得られたスラリーを遠心 （ 6 0 0 X g、 5分間、 4 °C) してべ レッ トを得た。 得られたペレッ トを l m l の酵素溶液 （ 0. 0 1 %の collagenase/dispase ( Boehrmger Manheim 社製)、 1 0 0 U m 1 の benzylpenicillin potassium, 丄 0 0 i g /m l の streptomycin sulfate, 2 0 U /m 1 の deoxyribonuclease I、 0. 1 4 7 x gノ m 1 の tosyl- lysine-chloromethylketone を添加した H B S S ) に懸濁し、 振盪を加 The cerebrum was isolated from the transgenic rat (1 animal) into which the large T antigen gene of the temperature-sensitive mutant tsA58 of SV40 obtained in Example 1 was introduced. The cerebrum excised in a clean bench was ice-cooled with an adjustment buffer (10 mM Hepes, lOUZ ml of benzylpenicillin potassium, 100 / igzml of streptomycin sulfate, 0.5% ゥ serum albumin). after extensive washing with HBSS) containing, minced cerebral to l to 2 mm 3, were transferred to lm 1 for Taber type tape off b emissions made Homogenai Heather (WHEATON Inc.), for adjustment of cold 1 m 1 ice The buffer was added, and four up and down strokes were performed to homogenize the tissue to obtain a slurry. The obtained slurry was centrifuged (600 × g, 5 minutes, 4 ° C.) to obtain a pellet. The obtained pellet was treated with lml of enzyme solution (0.01% collagenase / dispase (Boehrmger Manheim), 100 Um1 of benzylpenicillin potassium, 丄 100 ig / ml of streptomycin sulfate, 20 UU / m 1 of deoxyribonuclease I and 0.147 xg of m 1 tosyl-lysine-chloromethylketone).
1 8
えた水浴中で酵素処理 （ 3 7 ：、 3 0分間） を行い、 不要な組織から毛 細血管を分離した。 遠心 （ 6 0 0 X g、 5分間、 4 °C) してペレッ トを 得た。 得られたペレッ 卜から不要な組織を除去するため、 1 0 m 1 の 1 6 %のデキス トランを含む H B S Sにペレッ トを懸濁し、 遠心 （ 1 , 0 0 0 X g , 1 5分間、 4 °C) により毛細血管画分のペレツ 卜を得た。 得 られたペレッ 卜を再び 1 m 1 の酵素溶液に懸濁して酵素処理 （ 3 7 °C、 3 0分間） を行うことで毛細血管を細切した。 遠心 （ 6 0 0 X g、 5分 間、 4 °C ) してペレッ トを得た。 次に、 得られたペレッ トを 2 m 1 の培 養液 ( 1 5 // g / 1 の endothelial cell growth factor, 1 0 0 U /m 1 の benzylpenicillin potassium、 1 0 0 x gZm l の streptomycin sulfate, 2 . 5 0 g Zm 1 の amphotericin Bを添加した D M E M ) に分散して 1枚のコラーゲンタイプ I をコートした 3 5 mm Φ培養シャ ーレ一 （Becton Dickinson社製） に播種した。 3 3 °Cの炭酸ガス培養器 ( 5 C 02 - 9 5 % A i r、 飽和湿度） 内で培養 （初代培養） した。 培地を 1週間に 2回交換し、 継代はトリプシン液 （ 0. 0 5 %の Trypsin、1 8 Enzyme treatment (37 :, 30 minutes) was performed in the obtained water bath to separate capillaries from unnecessary tissues. The pellet was obtained by centrifugation (600 × g, 5 minutes, 4 ° C.). To remove unnecessary tissue from the resulting pellet, suspend the pellet in 10 ml of HBSS containing 16% dextran, and centrifuge (1, 000 Xg, 15 minutes, 4 hours). ° C) to obtain a pellet of the capillary fraction. The obtained pellet was suspended again in a 1 ml enzyme solution and subjected to enzyme treatment (37 ° C, 30 minutes) to cut the capillaries. The pellet was obtained by centrifugation (600 × g, 5 minutes, 4 ° C.). Next, the obtained pellet was added to 2 ml of a culture solution (15 // g / 1 endothelial cell growth factor, 100 U / m1 of benzylpenicillin potassium, 100 x gZml of streptomycin sulfate). , 2.5 μg Zm 1 amphotericin B) and dispersed in DMEM), and seeded on one 35 mm φ culture dish (Becton Dickinson) coated with one collagen type I. 3 3 ° C carbon dioxide incubator at (5 C 0 2 - 9 5 % A ir, saturated humidity) and incubated in a (primary culture). The medium was changed twice a week, and the passage was performed using trypsin solution (0.05% Trypsin,
0. 5 3 mM E D T A ； Gibco BRL社製） を用いて細胞を剥離し、 細胞 を分散播種した。 継代はおよそ 1週間隔で行った。 3回の継代の後、 1 0 2〜 1 0 3個の細胞をコラーゲンタイプ I をコートした 1 0 Ο πιιηφ培 養シャーレ一 （Becton Dickinson社製） に播種した。 3 3 °Cの炭酸ガス 培養器内で培養してコロニー形成を行った。培地を 1週間に 2回交換し、 7〜 1 0 日後にコロニーを形成した増殖速度の比較的速いコロニ一をべ ニシリ ンカップを用いて周囲の細胞から単離し、 得られた細胞を再び 1 0 0 mm (/ 培養シャーレ一に播種して 3 3 °Cの炭酸ガス培養器内で培養 してコロニー形成を行った。 ペニシリ ンカップを用いて増殖速度の比較 的速いコロニーを周囲の細胞から単離して 5種の細胞株 （T R— B B BThe cells were detached using 0.5 mM EDTA (manufactured by Gibco BRL) and dispersed and seeded. Passaging was performed at approximately weekly intervals. After three passages, were seeded in 1 0 2 to 1 0 3 cells were coated with collagen type I 1 0 Ο πιιηφ culture dish one (Becton Dickinson Co., Ltd.). Colonies were formed by culturing in a carbon dioxide incubator at 33 ° C. The medium was changed twice a week, colonies that formed colonies at a relatively high growth rate after 7 to 10 days were isolated from surrounding cells using a vesicinate cup, and the obtained cells were recovered again. 0 mm (/ inoculated in a culture dish and cultured in a carbon dioxide incubator at 33 ° C to form colonies. Using a penicillin cup, colonies with a relatively high growth rate were isolated from surrounding cells. 5 cell lines (TR-BBB
1 、 T R— B B B 5 、 T R— B B B 6 、 T R— B B B 1 1 、 T R — B B 1, TR—BBB5, TR—BBB6, TR—BBBB1,1, TR—BB
1 9
B 1 3 ) を得た。 これらの細胞株は内皮細胞特異的なスピン ドルフアイ バー状の継代を示した。 なお、 T R— B B B 1 3株は、 ブタペス ト条約 に基づいて日本通商産業省工業技術院生命工学工業技術研究所に受託番 号 F E P M B P— 6 8 7 3 として寄託されている。 1 9 B 13) was obtained. These cell lines showed endothelial cell-specific spindol fiber passages. The TR-BBB13 strain has been deposited under the Budapest Treaty with the Ministry of Economy, Trade and Industry of Japan, as a deposit number FEPMBP-66873 at the Research Institute of Biotechnology and Industrial Technology.
2 - 2 (ラージ T抗原タンパク質の確認） 2-2 (Confirmation of large T antigen protein)
上記実施例 2 - 1で得られた 5種の細胞株におけるラージ T抗原蛋白 質の発現をウェスタンブロッ ト法 （実験医学別冊バイオマニュアル U P シリーズ 「分子生物学的アプローチによる癌研究プロ トコール」 1 0 8 〜 1 1 5頁、 羊土社、 1 9 9 5年発行） により検討した。 5種の細胞株 (継代数 ： 2 0 ) を 9 0 πιπιφ培養シャーレ一で飽和まで培養した。 回 収した細胞を 3 % S D S— P B S ( p H 7.4 )で可溶化した後、遠心（ 1 0， 0 0 0 r p m 1 0分間） して不溶画分を除去した後、 ブラッ ドフ ォ一ド法 （BIO-RAD社製プロテインアツセィキッ ト II を使用） で総蛋 白質量を定量した。 それぞれ 2 O gの蛋白質を S D Sポリアクリルァ ミ ドゲル電気泳動で分離後、 ニトロセルロース膜に転写した。 3 %スキ ムミルク溶液でブロッキングしたニトロセルロース膜に 1次抗体として 抗 S V 4 0ラージ T抗原抗体 （CALBIOCHEM社製、 DP02-C) を、 2 次抗体として H R P標識抗マウス I g G抗体 （Amersham社製） を反応 させ、 ラージ T抗原蛋白質特異的な反応をアマシャム社製 ECL ウェス 夕ンブロッテイ ング検出システム （RPN2106M1) を用いて検出した。 5種の細胞株全てにおいてラージ T抗原蛋白質の発現を確認した。 Expression of the large T antigen protein in the five cell lines obtained in Example 2-1 above was determined by Western blotting (Experimental Medicine Separate Volume Biomanual UP Series “Cancer Research Protocol by Molecular Biological Approach”). (8-115 pages, Yodosha, published in 1995). Five cell lines (passage number: 20) were cultured in a 90 πιπιφ culture dish until saturation. The collected cells are solubilized with 3% SDS-PBS (pH 7.4), centrifuged (10,000 rpm for 10 minutes) to remove insoluble fractions, and then subjected to a blood feed method. (Using a protein assay kit II manufactured by BIO-RAD) to quantify the total protein mass. After 2 Og of each protein was separated by SDS polyacrylamide gel electrophoresis, it was transferred to a nitrocellulose membrane. An anti-SV40 large T antigen antibody (CALBIOCHEM, DP02-C) was used as a primary antibody on the nitrocellulose membrane blocked with a 3% skim milk solution, and an HRP-labeled anti-mouse IgG antibody (Amersham) was used as a secondary antibody. And the large T antigen protein-specific reaction was detected using Amersham's ECL Wes' evening blotting detection system (RPN2106M1). Expression of the large T antigen protein was confirmed in all five cell lines.
2 - 3 (細胞の同定） 2-3 (cell identification)
上記実施例 2 - 1で得られた細胞株が脳毛細血管内皮細胞であること を、 G L UT - - 1輸送担体および p—糖蛋白質の発現をウェスタンプロ ッティ ング法で検定した。 得られた各細胞株について、 実施例 2— 2と 同じ方法で作製したニ トロセルロース膜を用いて、 1次抗体として抗マ The cell lines obtained in Example 2-1 above were brain capillary endothelial cells, and the expression of GLUT-1 transporter and p-glycoprotein was assayed by Western blotting. For each of the obtained cell lines, an anti-macro antibody was used as a primary antibody using a nitrocellulose membrane prepared in the same manner as in Example 2-2.
20
ウス G L UT— 1抗体 （Chemicon社製、 Temecular, CA) 又は抗 P _糖 蛋白質ゥサギ抗体 （抗 md r抗体、 Oncogene Research Products ¾S¾) を、 2次抗体として H R P標識扰マウス I g G抗体 （Amersham 社製） 又は HR P標識抗ゥサギ I g G抗体 （Cappel社製） を反応させ、 G L U T - 1 蛋白質あるいは p 糖蛋白質特異的な反応をアマシャム社製 ECLウェスタンブロッテイ ング検出システム （RPN2106M1) を用いて 検出した。 5種の細胞株全てにおいて G L UT - 1蛋白質及び p—糖蛋 白質の発現が確認された。 従って、 得られた 5種の細胞が脳毛細血管内 皮細胞であることが同定された。 20 Mouse GLUT-1 antibody (Chemicon, Temecular, CA) or anti-P_glycoprotein 蛋白 sagi antibody (anti-mdr antibody, Oncogene Research Products¾S¾), and HRP-labeled 扰 mouse IgG antibody (Amersham GLUT-1 protein or p-glycoprotein-specific reaction using the ACL's ECL Western Blotting Detection System (RPN2106M1) or HRP-labeled anti-Egret IgG antibody (Cappel). Detected. Expression of GLUT-1 protein and p-glycoprotein was confirmed in all five cell lines. Therefore, the obtained five types of cells were identified as brain capillary endothelial cells.
2 - 4 (グルコース輸送能の確認） 2-4 (Confirmation of glucose transport ability)
上記実施例 2 1で得られた細胞株 T R _ B B B 1、T R— B B B 5、 TR B B B 6、 T R B B B 1 1、 T R B B B 1 3が機能的な G L U T— 1輸送担体を持つことを、 3—〇MG (3-o-methyl-D-glucose) 取り込み能を測定し、 濃度依存的なグルコース輸送能を示すことで機能 的な G L UT— 1輸送担体を有することを確認した。 すなわち、 2 4穴 細胞培養用プレートに T R B B B株を 3 X I 05Zゥエル Zm l とな るように播き、 3 3 °Cの炭酸ガス培養基で 2 4時間培養して細胞をコン フルェン トにした。 3—〇M Gの取り込みの測定は次の要領で行った。 まず、 培地を吸引して除去した後、 3 7 °Cに温めた 2 3 2 k B Q Zm 1 の [3H] 3— O M Gを含む uptake bufferを 0. 2 m 1カロえた。 uptake buffer ( 1 2 2 mMの N a C 1 、 3 mMの K C 1 、 1. 4mMの C a C 1 2、 1. 4mMの M g S〇4 * 7 H2〇、 0. 4mMの K2H P 04、 1 O mMの H e p e s、 2 5 mMの N a H C 03) は、 5 % C〇 2— 9 5 % O 2で 2 0分間バブリ ングして N a〇 Hで p H 7. 4に調製したもので ある。 1 0秒後に uptake bufferを取り除き、 4 °Cの uptake bufferで 洗浄した。 以下、 uptake buffer を取り除く までの時間を 2 0秒間、 3 It was confirmed that the cell lines TR_BBB1, TR—BBB5, TRBBB6, TRBBB11, and TRBBB13 obtained in Example 21 described above had a functional GLUT-1 transport carrier. The ability to take up (3-o-methyl-D-glucose) was measured, and it was confirmed that it had a functional GLUT-1 transporter by showing a concentration-dependent glucose transport ability. That is, the TRBBB strain was seeded on a 24-well cell culture plate at a concentration of 3 XI 0 5 Z ゥ Zml, and cultured for 24 hours in a 33 ° C carbon dioxide gas culture medium to make the cells confluent. . The measurement of the uptake of 3- 3MG was performed as follows. First, after removing the medium was aspirated, was 0. 2 m 1 Karoe the uptake buffer containing 2 3 2 k BQ Zm of 1 [3 H] 3- OMG warming to 3 7 ° C. uptake buffer (1 2 2 mM of N a C 1, 3 mM of KC 1, 1. C a C 1 2 of 4 mM, 1. 4 mM of M g S_〇 4 * 7 H 2 〇 of 0. 4 mM K 2 HP 0 4, 1 O mM of H EPES, 2 5 mM of N a HC 0 3) 5% C_〇 2 - p H 7 with 9 5% O 2 with and Baburi ring 2 0 min N A_〇 H . Prepared in 4. After 10 seconds, the uptake buffer was removed and washed with a 4 ° C uptake buffer. Hereafter, the time to remove the uptake buffer is 20 seconds, 3
2 1
0秒間、 1分間として同様の操作を行った。 細胞を 1 %のトライ トン X 一 1 0 0を含む l m 1 の P B Sでー晚可溶化し、 液体シンチレーショ ン カウンターを用いて放射活性を測定し、 3 — OMGの取り込み能の直線 性を確認した。 結果、 2 0秒間の取り込み時間を設定した。 twenty one The same operation was performed for 0 minute and 1 minute. The cells were solubilized in 1 ml of PBS containing 1% Triton X-100 in lm 1 and the radioactivity was measured using a liquid scintillation counter to confirm the linearity of 3 — OMG uptake. did. As a result, a capture time of 20 seconds was set.
次に、 3 — OMGの取り込みの基質濃度依存性を検討した。 細胞を 3 7 °Cに温めた uptake bufferで洗浄した後、 3 7 °Cに温めた 4 6 2 k B (1 ゥェルの [31^] 3 —〇 M Gを含む uptake bufferを 0. 2 m l 加え た。 ただし、 非標識体の 3— OMGを 0、 0. 5、 1 . 5、 1 0、 2 0、 3 0、 5 0 mM含む uptake bufferを使用して 3 — OM Gの各濃度液と した。 2 0秒後に uptake bufferを取り除き、 4 の 1 0111 1の非標識 体 3 _〇MGを含む uptake bufferで洗浄した。 次に、 1 %のトライ ト ン X - 1 0 0を含む l m 1 の P B Sでー晚可溶化し、 液体シンチレーシ ョンカウンタ一を用いて放射活性を測定した。 なお、、 3 一 OMGの濃度 に対する取り込み速度のプロッ ト式 （V = Vm a x X [ S ] Z (Km + [ S ]) ； Vm a xは最大速度定数、 Kmはミカエリス定数、 [ S ] は基質 濃度） を用いて 3 — OMGの取り込みの Kmと Vm a xを非線形最小二 乗法プログラム (Yamaoka K. et al.， J. Pharmacobio-Dyn., 4, 879-885, 1981) を用いて解析した。 この結果、 G L UT— 1 の基質である [3H] 3 一 OMGの取り込みは濃度依存的であり、 そのミカエリス定数 （Km) は 5. 6 mM、 最大取り込み速度定数 （Vm a x ) は 4 5 n m o l /m i nノ mg p r o t e i nであった。 その初期取り込み速度を 7. 0 7〜 1 0. 2 l / m i n /m g p r o t e i nであった。 糸吉果を表 1 に示す。 Next, the dependence of 3 — OMG uptake on the substrate concentration was examined. After washing the cells with the uptake buffer warmed at 37 ° C, warm up to 37 ° C 4 62 kB (0.2 ml of uptake buffer containing 1 gel of [ 3 1 ^] 3 —〇 MG However, using an uptake buffer containing 0, 0.5, 1.5, 10, 20, 30 and 50 mM of unlabeled 3-OMG, each concentration solution of 3 — OMG was used. After 20 seconds, the uptake buffer was removed, and the plate was washed with an uptake buffer containing the unlabeled 3_ 1MG of 4 0111 1 in 4. Then, the lm containing 1% of Triton X-100 After solubilization with PBS in Step 1, radioactivity was measured using a liquid scintillation counter, and the plot expression of the uptake rate with respect to the concentration of 31 OMG (V = Vmax X [S] Z (Km + [S]); Vm ax is the maximum rate constant, Km is the Michaelis constant, [S] is the substrate concentration), and the Km and Vmax of the uptake of OMG are calculated using the nonlinear least squares program (Yamaoka K. et al. ., J. Pharmacobio-Dyn., 4, 8 79-885, 1981) As a result, the uptake of [ 3 H] 3 OMG, a substrate of GLUT-1, was concentration-dependent, and its Michaelis constant (Km) was 5.6 mM. The maximum uptake rate constant (Vmax) was 45 nmol / min / mg protein, and the initial uptake rate was 7.07-10.2 l / min / mg protein. Figure 1 shows.
22
表 1 細 胞 取り込み初速度 （ l/mi n/mg protei n) twenty two Table 1 Initial cell uptake rate (l / min / mg protein)
TR-BBB1 8.12±0.62 TR-BBB1 8.12 ± 0.62
TR-BBB5 10· 1 ± 1 · 32 TR-BBB5 10 1 ± 1 32
TR-BBB6 7.07±0.92 TR-BBB6 7.07 ± 0.92
TR-BBB11 10.2±0.62 TR-BBB11 10.2 ± 0.62
TR-BBB13 8.96±0.50 TR-BBB13 8.96 ± 0.50
2 - 5 (スカベンジャーレセプ夕一機能の確認） 2-5 (Confirmation of Scavenger Reception Yuichi function)
上記実施例 2 ― 1で得られた細胞株 T R— B B B 1 3が機能的なスカ ベンジャ ー レセプタ ー を持つ こ と を、 蛍光標識体であ る 1，1' - dioctadecyl- 3,3,3',3'-tetramethyl-indocarbocyanine perchlorate標識 ァセチリレイ匕： L D L (Dil-Ac-LDL, Biomedical Technologies, Stoughton, MA) の取り込みを測定することで解析した。 カバ一グラスに細胞株 T R— B B B 1 3を 1 X I 0 5Zゥエル Zm l 培地で播種し、 3 3 °Cの炭 酸ガス培養器内で 4 8時間培養して細胞をコンフルェントにした。 D i 1 一 A c — L D Lの取り込み測定は、 まず、 培地を吸引して除去した後 に予め 3 7 °Cに温めた uptake buffer II ( 1 2 2 mMの N a C l 、 3 mThe fact that the cell line TR-BBB13 obtained in Example 2-1 above has a functional scavenger receptor was confirmed by the use of the fluorescent label 1,1, '-dioctadecyl-3,3,3. ', 3'-Tetramethyl-indocarbocyanine perchlorate labeled acetyleylamide: Analyzed by measuring the incorporation of LDL (Dil-Ac-LDL, Biomedical Technologies, Stoughton, MA). Hippo seeded at a glass cell line TR- BBB 1 3 in 1 XI 0 5 Z Ueru Zm l medium, the cells were in Konfuruento cultured for 48 hours in a 3 3 ° carbon dioxide gas incubator of C. To measure the uptake of Di11-Ac-LDL, first remove the medium by aspiration and then warm up to 37 ° C in advance using uptake buffer II (122 mM NaCl, 3 m
Mの K C 1 、 1. 4 mMの C a C 1 1. 4 mMの M g S O 7 H O. 0. 4 mMの K 7 H P O O mMの H e p e s 、 2 5 mMの N aM KC 1, 1.4 mM Ca C 1 1.4 mM Mg SO 7 H O.0.4 mM K 7 HPOO mM Hepes, 25 mM Na
H C 03、 1 O mMの D— g 1 u c o s eの溶液を 5 % C〇 2— 9 5 % O 2で 2 0分間バブリ ングして、 N a OHで p H 7. 4に調整） で細胞を 洗浄した。 次に、 3 7 °Cに温めた 1 0 g / 2 0 0 / 1 の D i 1 — A c _ L D Lを含む uptake buffer II を 0. 2 m 1 加え 3 0分間炭酸ガス培 養器でィンキュベーショ ンした。 4時間後に uptake buffer II を除去し、 HC 0 3, 1 O mM of D- g 1 ucose solution of 5% C_〇 2 - 9 5% O 2 in and 2 0 min Baburi ring, cells p H 7. adjusted to 4) with N a OH Was washed. Next, add 0.2 ml of uptake buffer II containing 10 g / 200/1 Di 1 — Ac _ LDL warmed to 37 ° C, and incubate in a carbon dioxide incubator for 30 minutes. I did. After 4 hours, remove uptake buffer II,
23
4 °Cの uptake buffer IIで 3回洗浄した。 次に、 3 % formaldehyde/' P B Sを加え 2 0分間室温に保持して固定したものを共焦点レーザー顕 微鏡を用いて細胞内に取り込まれた蛍光を測定した。 その結果、 ス力べ ンジャ ー レセプ夕 一 の リ ガ ン ド であ る 1，1'- dioctadecyl-3，3，3'，3'- tetramethyl-mdocarbocyanine perchlorateで標 g哉されたァセチルイ匕 L D L (D i 1 — A c — L D L ) が細胞内に取り込まれていることを確認 した。 また、 他の細胞株でも同様の結果が得た。 twenty three Washed three times with uptake buffer II at 4 ° C. Next, 3% formaldehyde / 'PBS was added, and the mixture was fixed at room temperature for 20 minutes, and the fluorescence taken into the cells was measured using a confocal laser microscope. As a result, acetiluidani LDL, which was treated with 1,1'-dioctadecyl-3,3,3 ', 3'-tetramethyl-mdocarbocyanine perchlorate, which is one of the ligans of the Sengai Benger Reception Di 1 — Ac — LDL) was taken up into the cells. Similar results were obtained with other cell lines.
2 - 6 (アル力リフォスファターゼ及びァ一ダル夕ミルランスぺプチダ ーゼ活性の確認） 2-6 (Confirmation of the activity of alkaline phosphatase and amyloid peptidase)
上記実施例 2— 1で得られた細胞株が脳毛細血管内皮細胞に発現して いるアル力リフォスファ夕ーゼ活性及びァダル夕ミルトランスべプチダ ーゼ活性を発現することを常法に従い測定した。 測定には、 アルカリ性 ホスファ B—テス トヮコ一及びァ 一 GT P—テス トヮコ一 （和光純薬社 製） を使用し、 それぞれのキッ トに記載された標準測定法に従って測定 した。 尚、 タンパク質量は、 ブラッ ドフォード法 （プロテインアツセィ キッ ト II； BIO-RAD 社製） により測定した。 その結果、 アルカリフォ スファ夕一ゼ活性及びァーダル夕ミルトランスぺプチダーゼ活性はラッ ト脳毛細血管リ ッチ画分 （Brain Capillaries) を対照に、 それぞれ 8. 7〜 2 5. 8 %及び 5. 4〜 2 2. 6 %を示し、 脳毛細血管内皮細胞特 異酵素の発現を認めた。 結果を表 2に示す。 It was measured according to a standard method that the cell line obtained in the above Example 2-1 expresses the activity of lipophilic phosphatase and the activity of aldaramil transbeptidase expressed in brain capillary endothelial cells. . Alkaline phospha B-test and A-1 GTP-test (manufactured by Wako Pure Chemical Industries, Ltd.) were used for the measurement according to the standard measurement method described in each kit. The amount of protein was measured by the Bradford method (Protein Atsushi Kit II; manufactured by BIO-RAD). As a result, the alkaline phosphatase activity and the aardal mil transpeptidase activity were 8.7 to 25.8% and 5, respectively, based on the rat brain capillary rich fraction (Brain Capillaries). It showed 4 to 22.6%, indicating expression of brain capillary endothelial cell-specific enzyme. Table 2 shows the results.
24
表 2 twenty four Table 2
A L P活性 T - G Τ_Ρ活性 細 胞 μ U/mg prote i n (対照比 U/mg protei n (対照比％) ALP activity T-G Τ_Τ activity cells μ U / mg prote inn (control ratio U / mg protein (control ratio%)
TR-BBB1 23.7±7.17(25.8¾) 3.62±0.47(12.4¾)TR-BBB1 23.7 ± 7.17 (25.8¾) 3.62 ± 0.47 (12.4¾)
TR-BBB5 11.9±2.92 (13.0%) 2.05±0.76( 7.0¾)TR-BBB5 11.9 ± 2.92 (13.0%) 2.05 ± 0.76 (7.0¾)
TR-BBB6 22.3± 8.78 (24.3¾) 1.58土 0.52 ( 5.4¾)TR-BBB6 22.3 ± 8.78 (24.3¾) 1.58 Sat 0.52 (5.4¾)
TR-BBB11 8.05±2.37( 8.7%) 6.60± 0.93 (22.6%)TR-BBB11 8.05 ± 2.37 (8.7%) 6.60 ± 0.93 (22.6%)
TR-BBB13 13.7±3· 92(14.9%) 5.60±1.08(19.1%) 対照 TR-BBB13 13.7 ± 3 92 (14.9%) 5.60 ± 1.08 (19.1%) Control
9 8±30.8(100%) 29.2±Π.8(100 ) 9 8 ± 30.8 (100%) 29.2 ± Π.8 (100)
(B ra i nCap i i es) 実施例 3 (ァス トロサイ ト細胞株の調製） (BrainCaps) Example 3 (Preparation of Astrosite Cell Line)
3— 1 (ァス トロサイ ト細胞株の分離） 3-1 (Astrocyte cell line isolation)
上記の実施例 1で得られた S V 4 0の温度感受性突然変異株 t s A 5 8のラージ T抗原遺伝子を導入したトランスジエニックラッ ト （ 5匹） から大脳を摘出した。 脳ァス トロサイ ト細胞の分離と回収は酵素法を用 いて以下のように行った。 大脳を 1 2 1 °C、 1 5分間で滅菌し、 分離バ ッファー （ 1 2 2 mMの N a C l 、 3 mMの K C 1 、 1. 4 mMの C a C l 2、 1. 2 mMの M g S 04、 0. 4mMの K2H P 04、 1 0 m M の G l u c o s e、 l O mMの H e p e s ; p H 7. 4) の入ったシャ ーレに入れてクリーンベンチ内に移し、 分離バッファ一で洗浄した。 大 脳をはさみで 2 mm角に細く し、 あるいは小さく刻み、 1 m l の 2. 4 UZm 1 のディスパ一ゼ溶液を加え、 3 7 °C、 1 0分間で酵素処理した。 未消化断片を除去後、 培地 （ 1 0 0 UZm 1 の Penicillin G、 1 0 0 m g Z m 1 の streptomycin sulfate、 1 0 %の fetal bovine serum, DM E M 9. 6 gを 2回蒸留水で 1 Lにしたもの） に懸濁 The cerebrum was isolated from the transgenic rats (5) into which the large T antigen gene of the temperature-sensitive mutant tsA58 of SV40 obtained in Example 1 was introduced. Separation and recovery of brain astrocyte cells were performed using the enzymatic method as follows. Cerebral sterilized at 1 2 1 ° C, 1 5 min, separation bar Ffa (1 2 2 mM of N a C l, 3 mM of KC 1, 1. 4 mM of C a C l 2, 1. 2 mM of M g S 0 4, 0. 4mM of K 2 HP 0 4, 1 0 m M of G lucose, l O mM of H epes; p H 7. 4) of contained within a clean bench placed in a petri Les And washed with a separation buffer. The cerebrum was cut into 2 mm squares or minced with scissors, and 1 ml of 2.4 UZm1 dispase solution was added, followed by enzyme treatment at 37 ° C for 10 minutes. After removing undigested fragments, culture medium (100 UZm 1 of Penicillin G, 100 mg of Zm 1 of streptomycin sulfate, 10% of fetal bovine serum, 9.6 g of DEM Suspended in L)
25
得られた細胞懸濁液を培養皿 （Cornmg社製 ； # 4 3 0 1 6 7 ) にて 3 7 °Cの 5 % C〇 2イ ンキュベーターで 2 日間培養した。 3 日 目に 3 3 °Cの 5 % C 02インキュベーターに移して培養を継続した。 細胞の継 代には 0. 1 %のコラゲナーゼ Zデイスパーゼ溶液で 3 7 °C、 5分間処 理した後、 4°Cで 2時間放置して細胞を培養皿から剥離した。 この細胞 のクローニングはコロニー形成法で行った。 1〜 3回継代した細胞につ いて、 細胞を剥離後、 1 02〜 1 03個の細胞を 9 0 mm培養皿に播き、 コロニー形成を行った。 培地を吸引除去後、 片側にグリースを塗ったク ローニング力ップをコロニーが中央にくるように培養皿に立てた。 力ッ プ内にのみ 0. 2 5 %のトリプシン— E D TA液を加え、 3 3 ° で 1分 間インキュベートした。 培地をカップ内に加え、 ピペッティ ングして細 胞を剥し、 ダブリ ングタイムが約 2 4時間で増殖し、 約 3 0代まで継代 が進んでも増殖性に顕著な変化は見られなく安定であった。 これらを T R - A S T 3 2、 T R -A S T 8 1 1 , TR -A S T 9 1 2 T R— A S T 9 3 2、 T R— A S T 9 4 3株とそれぞれ命名した。 なお、 前記の ように、 T R— A S T 9 3 2株は、 ブタペス ト条約に基づいて日本通商 産業省工業技術院生命工学工業技術研究所に受託番号 F Ε ΡΜ Β Ρ - 6 2 8 3として寄託されている。 twenty five The obtained cell suspension was cultured in a 5% C 培養2 incubator at 37 ° C for 2 days in a culture dish (Cornmg; # 43016). On day 3 3 3 ° and transferred to 5% C 0 2 incubator C and the culture was continued. For passage of the cells, the cells were treated with a 0.1% collagenase Z dispase solution at 37 ° C for 5 minutes, and then left at 4 ° C for 2 hours to detach the cells from the culture dish. Cloning of the cells was performed by the colony forming method. 1-3 times have passaged cells Nitsu, after peeling off the cells were plated 1 0 2 to 1 0 3 cells into 9 0 mm culture dish, were colony formation. After removing the medium by suction, a cloning force greased on one side was placed on a culture dish such that the colony was at the center. 0.25% trypsin-EDTA solution was added only in the forceps and incubated at 33 ° for 1 minute. The medium was added to the cup, and the cells were detached by pipetting.The cells grew in a doubling time of about 24 hours. Was. These were named TR-AST32, TR-AST811 and TR-AST912 TR-AST933 and TR-AST943 strains, respectively. As described above, TR-AST932 strain was deposited under the Budapest Treaty with the Nippon International Trade and Industry Institute of Industrial Science and Technology, Institute of Biotechnology and Industrial Technology under the accession number F Ε ΡΜ Β Ρ-62883. Have been.
3 - 2 (樹立した株化ァス トロサイ ト細胞の G F APの発現と Lダル夕 ミン酸輸送能） 3-2 (GFAP expression and L-dalminic acid transport capacity of established established astrocyte cells)
実施例 3— 1で得られた細胞株 T R _A S T 3 2、 T R— A S T 8 1 1 , T R -A S T 9 1 2 , T R— A S T 9 3 2、 TR A S T 9 4 3の ァス トロサイ ト特有の蛋白質である glial fibrillary acidic Protein ( G F A P ) の発現を免疫染色によって検討した。 細胞をカバーグラス上に 培養しサンプルとした。 P L P溶液 （過塩素酸ナトリウム · Lリジン塩 酸塩 · パラホルムアルデヒ ド混合液） で氷冷下で 1 5分間固定後、 プロ Astrosite-specific cell lines TR_A ST32, TR-AST 811, TR-AST 912, TR-AST 932 and TR AST 943 obtained in Example 3-1 The expression of glial fibrillary acidic protein (GFAP), a protein, was examined by immunostaining. The cells were cultured on a cover glass to obtain a sample. Fix with a PLP solution (a mixture of sodium perchlorate, L-lysine hydrochloride and paraformaldehyde) for 15 minutes under ice-cooling.
26
ッキング溶液を用いてブロッキングを室温で 6 0分間行った。 1 0 0倍 希釈した抗 G F A P坊体を室温で 6 0分作用させた後、 リ ンスバッファ 一で 3回洗浄し、 5 0 0倍希釈したペルォキシターゼ標識抗ラビッ 卜 I g Gを 6 0分間作用させた。 サンプルをリ ンスバッファ一で十分に洗浄 後、 発色反応を行った。 氷冷した発色液を加え、 顕微鏡で観察し、 サン プルが発色した時点で冷 P B Sを加え反応を停止させ、 この時点でネガ ティブコントロールと発色の強さを比較した。 発色試薬として 3 0 %過 酸化水素含有 3， 3 - d i am i n o b e n z i d i n e - P B S溶液 を用いた。 その結果、 TR— A S T 3 2、 T R -A S T 8 1 1 , TR— A S T 9 1 2 , TR -A S T 9 3 2 , T R _ A S T 9 4 3株のいずれに おいても、 G F A Pの発現を示す要請の染色が確認された。 よって T R 一 A S T 3 2、 TR - A S T 8 1 1 , T R - A S T 9 1 2、 TR - A S T 9 3 2、 T R -A S T 9 4 3株はァス ト口サイ ト細胞株であることが 確認された。 26 Blocking was performed for 60 minutes at room temperature using the locking solution. After incubating the anti-GFAP colonies diluted 100 times at room temperature for 60 minutes, the cells were washed three times with rinse buffer, and the peroxidase-labeled anti-rabbit IgG diluted 1: 500 was acted on for 60 minutes. I let it. After thoroughly washing the sample with a rinse buffer, a color reaction was performed. An ice-cooled coloring solution was added, and the mixture was observed under a microscope. When the sample developed color, the reaction was stopped by adding cold PBS. At this time, the intensity of the coloring was compared with that of the negative control. A 3,3-diam inobenzidine-PBS solution containing 30% hydrogen peroxide was used as a coloring reagent. As a result, in all of the TR-AST32, TR-AST811 and TR-AST912, TR-AST932 and TR_AST943 strains, there was a request to show the expression of GFAP. Staining was confirmed. Therefore, it was confirmed that TR-1 AST32, TR-AST811, TR-AST910, TR-AST932, and TR-AST944 were strains of fast mouth cell lines. Was.
実施例 3— 1で得られた細胞株 T R— A S Τ 3 2、 T R— A S T 8 1 1， T R - A S T 9 1 2、 T R - A S T 9 3 2、 T R - A S T 9 4 3株 のグルタミ ン酸輸送能を、 [3 H] L— g u 1 u t a m i c a c i d ([3H] L一 G l u) の取り込みによって検討した。 2 4穴細胞培養用 プレー 卜に T R— A S T株を 2 X 1 05細胞/ゥエル培地となるように 播き、 3 3 °Cの C 02インキュべ一夕一で 3 0時間培養し、 コンフルェ ントにした。 [3 H] L— G 1 uの取り込みの測定は以下のように行った, まず培地を吸引し、 3 7 °Cに温めた上記 uptake buffer IIで洗浄後、 3 7 °Cに温めた 4 6. 3 k B q Zゥエルの [3H] L _ G l uと 9. 2 5 k B q /m l の [1 4 C] ィヌ リ ンを含む 0. 2 m 1 の uptake buffer II を添加し、 2、 5、 1 0、 3 0分後に トレーサー液を取り除いて 4 °Cの 1 m 1 の uptake buffer IIで 3回洗浄した。 これに 0. 7 5 m 1 の 1 % Glutamate of the cell strains TR—ASΤ32, TR—AST811, TR-AST910, TR-AST932, and TR-AST943 obtained in Example 3-1 Transport capacity was examined by incorporation of [ 3 H] L—gu 1 utamicacid ([ 3 H] L-G lu). 2 4 were seeded in well cell culture play Bok TR- AST lines such that 2 X 1 0 5 cells / Ueru medium, 3 3 ° C C 0 2 and incubator base Isseki culture one 3 0 hours, Konfurue I did. [ 3 H] L—G 1 u incorporation was measured as follows. First, the medium was aspirated, washed with the above uptake buffer II warmed to 37 ° C, and then heated to 37 ° C 4 6. Add 0.2 ml uptake buffer II containing [ 3 H] L _ Glu of 6.3 kB q Z ゥ L and [ 14 C] inulin of 9.25 kBq / ml. After 2, 5, 10, and 30 minutes, the tracer solution was removed, and the plate was washed three times with 1 ml of uptake buffer II at 4 ° C. 1% of 0.75 m 1
27
トライ トン X— 1 0 0溶液を加え、 一晩放置し、 細胞を可溶化し、 液体 シンチレーショ ンカウンターを用いて放射活性を測定した。 その結果、27 A Triton X-100 solution was added, the mixture was allowed to stand overnight, the cells were solubilized, and the radioactivity was measured using a liquid scintillation counter. as a result,
L—グルタミン酸トランスボー夕一の基質である [3H] L— G l uの 取り込みは濃度依存的であり、 そのミカエリス定数 （Km) は 9 6 M、 最大取り込み速度定数（Vm a x) は 1. 8 nm o l Zm i n /m g p r o t e i nであった。 この取り込みは N a +— f r e e b u f f e rで有意に阻害され、 また他の基質である L一 a s p a r t i c a c i dや D - a s p a r t i c a c i dで、 有意な阻害を示した。 結果 を表 3に示す。 表 3から、 得られた細胞株がァス トロサイ ト本来の機能 を保持していることが確認された。 It is L- glutamate transporter Bo evening one substrate [3 H] L- G lu uptake is concentration-dependent, the Michaelis constant (Km) is 9 6 M, the maximum uptake rate constant (Vm ax) is 1. It was 8 nmol Zmin / mg protein. This uptake was significantly inhibited by Na +-freebuffer, and was significantly inhibited by other substrates such as L-asparticacid and D-asparticacid. Table 3 shows the results. From Table 3, it was confirmed that the obtained cell line retained the original function of astrocyte.
表 3
Table 3
コン卜ロールに対する％で表記 実施例 4 (脳毛細血管周皮細胞の調製） Example 4 (Preparation of brain capillary pericytes)
4一 1 (脳毛細血管周皮細胞の分離） 4-1 (Separation of brain capillary pericytes)
大脳か ら の脳毛細血管周皮細胞の分離は Ichikawa ら の方法 Isolation of brain capillary pericytes from the cerebrum was performed by the method of Ichikawa et al.
(Ichikawa N et al" (1996) J. Pharmacol. Toxicol. Method., 36, 45-52) . Capetandes らの方法 (Capetandes A and Gerristen ME (1990) Invest. Ophthalmol. Vis. Sci., 31, 1738-1744) を改良して行った。 実施例 1で 得られた S V 4 0の温度感受性突然変異株 t s A 5 8のラージ T抗原遺 伝子を導入したトランスジエニックラッ ト （ 1 匹） から脳を摘出した。 摘出した脳は、 大脳以外の間脳、 脳管などを取り除いた後、 ク リーンべ ンチ内で氷冷した調製用緩衝液 （ 1 2 2 mMの N a C 1 、 3 mMの K C(Ichikawa N et al "(1996) J. Pharmacol. Toxicol. Method., 36, 45-52). Capetandes et al. (Capetandes A and Gerristen ME (1990) Invest. Ophthalmol. Vis. Sci., 31, 1738. -1744) from a transgenic rat (1 animal) into which the large T antigen gene of the temperature-sensitive mutant tsA58 of SV40 obtained in Example 1 was introduced. The removed brain was prepared by removing the mesencephalon, cerebral duct, etc. other than the cerebrum, and then cooling it in a clean bench with ice-cold preparation buffer (122 mM NaC1, 3 mM The KC
1 、 1. 4 m Mの C a C し'、 1 . 2 m Mの M g S O 4 · 7 H .，〇、 0. 1, 1. C a C of 4 m M ', 1. 2 m M of M g SO 4 · 7 H. , 〇, 0.
28
4 mMの K 2H P 04 、 1 0 m λΐの G l u c o s e 、 l O mMの H e p e s ； p H 7 . 4 ) でよく洗浄した。 洗浄した大脳は、 組織を 1 〜 2 m m 2に細切した。 細切した組織を 1 0 m 1 用 Potter-Elvehjem ホモゲナイ ザ一 （東京理化器械社製） に移し、 大脳の 4倍量の氷冷した調製用緩衝 液を加え、 1 0回のアップダウンのス トロークを行い組織をホモゲナイ ズし、 3 2 %のデキス トランを含む P B Sを等量加え、 3回のアップダ ゥンのス トロ一クを行いスラリーを得た。 28 4 mM of K 2 HP 0 4, 1 0 m λΐ of G lucose, l O mM of H EPES; washed well with p H 7 4).. The washed cerebrum was minced into 1-2 mm 2 tissue. Transfer the minced tissue to a Potter-Elvehjem homogenizer for 10 ml (manufactured by Tokyo Rikakikai Co., Ltd.), add 4 volumes of ice-cold preparation buffer of the cerebrum, and perform 10 times of up-down. A tissue was homogenized by troking, an equal volume of PBS containing 32% dextran was added, and a stroke of up-down was performed three times to obtain a slurry.
このスリ ラーを遠心 （ 4 °C下、 4 5 0 0 X gで 1 5分間） して得られ るペレッ トを 2 . 4 m l の酵素溶液 [ 0 . 0 6 6 %の collagenase/dispase (Boehringer Mannheim 社製）、 0 . 0 3 3 %の 8 八 （シグマ社製） を含む P B S ] に懸濁し、 振盪を加えた水溶中で酵素処理 （ 3 7 °C、 3 時間） を行い、 不要な細胞外マトリ ックスを分離し、 遠心 （ 4 °C下、 6 0 0 X gで 5分間） してペレッ トを得た。 得られたペレッ トを 1 0 m 1 の培 液 ( 1 0 0 /m 1 の benzylpenicillin potassium、 1 0 0 / g z m 1 の streDtomycin sulfate 2 . 5 0 /i g Zm l の ampnotericm B、 2 0 %の F C Sを添加した D M E M) に分散して 4枚の 3 5 mm <i)培養 シャーレ （Falcon社製） に種播した。 3 3 °Cの炭酸ガス培養器 （ 5 %の C〇 2 _ 9 5 %の A i r、 飽和湿度） 内で培養 （初代培養） した。 培地 を 1週間に 2回交換し、 継代はトリプシン液 （ 0 . 0 5 %の Trypsin、 0. 5 3 mMの E D T A ； Gibco BRL社製） を用いておよそ 1週間隔で 行った。 2回の継代の後、 1 0 4個の細胞を 1 0 0 m m φ培養シャーレ (Falcon社製） に種播した。 培地を 1週間に 2回交換し、 7 〜 1 0 日後 にコロニーを形成した増殖速度の比較的速いコロニーをペニシリ ンカツ プを用いて周囲の細胞から単離し、 得られた 1 0 3個の細胞を再び 1 0 0 mm φ培養シャーレに種播して 3 3 °Cの炭酸ガス培養器内で培養して コロニー形成を行った。 ベニシリ ンカップを用いて増殖速度の比較的速 The pellet obtained by centrifuging the chiller (4500 xg at 4 ° C for 15 minutes) was added to 2.4 ml of the enzyme solution [0.066% collagenase / dispase (Boehringer Suspended in PBS containing 0.033% of 88 (manufactured by Sigma)], and subjected to enzyme treatment (37 ° C, 3 hours) in an aqueous solution with shaking. The extracellular matrix was separated and centrifuged (600 xg at 4 ° C for 5 minutes) to obtain a pellet. The obtained pellet was added to a 10 ml culture medium (100 / m1 benzylpenicillin potassium, 100 / gzm1 streDtomycin sulfate 2.5.50 / ig Zm1 ampnotericm B, 20% FCS Was dispersed in DMEM) and seeded on four 35 mm <i) culture dishes (Falcon). 3 3 ° C CO 2 incubator (5% of the C_〇 2 _ 95% of the A ir, saturated humidity) and incubated in a (primary culture). The medium was changed twice a week, and subculture was performed at approximately one week intervals using a trypsin solution (0.05% Trypsin, 0.53 mM EDTA; Gibco BRL). After two passages were Tane播1 0 4 cells 1 0 0 mm phi culture plate (Falcon). Replace medium twice a week, 7 to isolated from the surrounding cells with a penicillin Nkatsu flop colonies with relatively high growth rate of forming the colonies after 1 0 days, 1 0 3 cells obtained Was again seeded on a 100 mm φ culture dish, and cultured in a carbon dioxide incubator at 33 ° C. to form a colony. The growth rate is relatively high using the Benicillin cup.
2 9
いコロニーを周囲の細胞から単離して 2種の細胞株 （T R— P C T 1 ， T R— P C T 2 ) を得た。 なお、 前記のように、 T R — P C T 1株は、 ブ夕ぺス ト条約に基づいて日本通商産業省工業技術院生命工学工業技術 研究所に受託番号 F E P M B P — 7 0 2 4として寄託されている。 4 - 2 (ラージ T抗原タンパク質の確認） 2 9 Colonies were isolated from surrounding cells to obtain two cell lines (TR-PCT1, TR-PCT2). As described above, one TR — PCT strain has been deposited with the Nippon International Trade and Industry Institute of Industrial Science and Technology, Institute of Biotechnology, Industrial Technology under the Busyeast Treaty under the accession number FEPMBP — 7204. . 4-2 (Confirmation of large T antigen protein)
実施例 4 一 1で得られた 2種の細胞株のラージ T抗原蛋白質を、 実施 例 2 — 2に記載のウエスタンプロッ ト法により検出した。 すなわち、 2 種の細胞株をそれぞれ P B Sで洗浄後、 l mLの可溶化溶液 （ 1 %の D S、 1 0 mMの T r i s 、 I mMの E D T A、 1 0 %の glycerin) で 可溶化し、 1 0 0 °Cで 1 0分間加熱した後、 遠心 （ 1 0 0 0 0 r p mで 1 0分間） して不溶画分を除去した後、 ブラッ ドフォード法 （ P I E R C E社製 B C Aプロテインアツセィ試薬 Aを使用） で総蛋白質量を定量 した。 それぞれ 1 0 /x gの蛋白質を S D Sポリアクリルアミ ドゲル電気 泳動で分離後、 ニトロセルロース膜に転写した。 3 %のスキムミルク溶 液でブロッキングしたニトロセルロース膜に 1次抗体として抗 S V 4 0 ラージ T抗原マウス抗体 （D P 0 2 — C ； CALBIOCHEM社製） を、 2 次抗体として H R P標識抗マウス I g G抗体 （Amersham社製） を反応 させ、 ラージ T抗原特異的な反応をアマシャム社製 E C Lウェス夕ンブ 口ティ ング検出システム （R P N 2 1 0 6 M 1 ) を用いて検出した。 結 果を表 6に示す。 この結果、 得られた 2種の細胞株全てにおいてラージ T抗原蛋白質を確認した。 Example 4 Large T antigen proteins of the two cell lines obtained in 11 were detected by the Western plot method described in Example 2-2. That is, each of the two cell lines was washed with PBS, and then solubilized with 1 mL of a solubilization solution (1% DS, 10 mM Tris, ImM EDTA, and 10% glycerin). After heating at 100 ° C for 10 minutes, centrifugation (100 rpm at 10 minutes) was performed to remove insoluble fractions, and then the Bradford method (BCA protein assay reagent A manufactured by PIERCE) was used. Used) to determine the total protein content. After 10 / xg of each protein was separated by SDS polyacrylamide gel electrophoresis, it was transferred to a nitrocellulose membrane. Anti-SV40 large T antigen mouse antibody (DP02-C; manufactured by CALBIOCHEM) was used as the primary antibody on the nitrocellulose membrane blocked with 3% skim milk solution, and HRP-labeled anti-mouse IgG was used as the secondary antibody. An antibody (Amersham) was allowed to react, and a large T antigen-specific reaction was detected using an ECL Wessling detection system (RPN210M1) manufactured by Amersham. Table 6 shows the results. As a result, large T antigen protein was confirmed in all of the obtained two cell lines.
4 - 3 ( P D G F受容体 /3 と T h y - 1 の確認） 4-3 (confirmation of PDGF receptor / 3 and Thy-1)
得られた細胞株を単層培養し、 細胞膜に発現された P D G F受容体 )3 と T h y — 1 を免疫染色し、 顕微鏡を用いて確認した。 実施例 4 一 1で 得られた細胞株 T R — P C T 1 を 24 well dish (Falcon社製） のカバ一 グラス上で培養した。 培養液を除去し、 細胞を P B Sで洗浄した後、 0 . The obtained cell line was cultured in a monolayer, immunostained for PDGF receptor) 3 and Thy-1 expressed on the cell membrane, and confirmed using a microscope. Example 4 The cell line T R —PCT 1 obtained in 11 was cultured on a cover glass of a 24-well dish (Falcon). After removing the culture medium and washing the cells with PBS,
3 0
5 mLの固定液 （ 3. 6 %のパラホルムアルデヒ ド） を加え、 4 °Cで 2 0分間放置後、 P B Sでよく洗浄した。 0. 5 mLの 3 % B S A— P B Sを加え、 室温で 2時間放置してブロッキングした後、 1次抗体 G P D G F受容体 βャギ抗体： Santacruze社製、 F I T C標識抗 T h y - 1 マウス抗体 ； Pharmingen社製） を室温で 1時間反応させた。 0. 1 % の B S A— P B Sで 5回洗浄後、 2次抗体（F I T C標識抗ャギ I g G ； シグマ社製 （P D G F受容体 検出のみ使用）） を室温で 1時間反応させ、 0. 1 %の B S A— P B Sで 5回洗浄した。 最後に標識した細胞をダリ セリ ン封入液 ( 9 0 %の glycerol、 1 m g Z m Lの p-phenylendiamine、 0. 0 1 Mの N a 2 H P 04 ; p H 8. 5 ) で封入した。 観察は顕微鏡で 行った。 この結果、 細胞株 T R— P C T 1では細胞膜上に P D G F受容 体 /3、 及び T h y - 1 の発現を認めた。 また P D G F受容体 /3の発現は、 ウエスタンプロッ ト、 R T— P C Rにおいても確認した。 なお、 T R— P C T 2についても同様の結果が得られた。 3 0 5 mL of fixative (3.6% paraformaldehyde) was added, left at 4 ° C for 20 minutes, and washed well with PBS. After adding 0.5 mL of 3% BSA-PBS and leaving at room temperature for 2 hours for blocking, the primary antibody GPDGF receptor β-goat antibody: Santacruze, FITC-labeled anti-Thy-1 mouse antibody; Pharmingen Was reacted at room temperature for 1 hour. After washing 5 times with 0.1% BSA-PBS, a secondary antibody (FITC-labeled anti-goat IgG; Sigma (PDGF receptor detection only)) was reacted for 1 hour at room temperature. Washed 5 times with% BSA-PBS. Finally labeled cells Dali serine fill fluid (90% of glycerol, 1 mg Z m L of p-phenylendiamine, of 0. 0 1 M N a 2 HP 0 4; p H 8. 5) was encapsulated in . Observation was performed with a microscope. As a result, in the cell line TR-PCT1, expression of PDGF receptor / 3 and Thy-1 was recognized on the cell membrane. The expression of PDGF receptor / 3 was also confirmed by Western plot and RT-PCR. Similar results were obtained for TR-PCT2.
4 - 4 (アンギオポェチン一 1 とォステオポェチン、 I C AM— 1 の確 、 / 4-4 (Angiopoetin 1 and Osteopoetin, confirm ICAM-1
上記実施例 4一 1で得られた細胞株を単層培養し、 細胞に発現された アンギオポェチン一 1 とォステオポェチン （Osteopontin)、 I C AM— 1 を R T— P C Rにて検出した。 すなわち、 細胞株 T R— P C T 1 を 1 0 0 mm φ培養シャーレ （Falcon社製） に培養した。 培養液を除去し、 細胞を P B Sで洗浄した後、 セルスクレーパー （IWAKI社製） にて細胞 をかき集め、 R NA抽出試薬 （Trizol； Gibco 社製） にて全 R NAを抽 出した。 逆転写酵素 （Rev Tra Ace： TOYOBO社製） を用いて、 抽出し た全 R NA l ^ gから c D NAを合成し、 P C R法 （ e x T a cj ； 宝酒 造社製） にて発現を確認した。 P C R増幅産物の確認は、 5 %アク リル アミ ドゲルの電気泳動にて行った。 この結果、 細胞株 T R— P C T 1 に The cell line obtained in Example 4-11 was cultured in a monolayer, and angiopoetin-11, osteopontin, and ICAM-1 expressed in the cells were detected by RT-PCR. That is, the cell line TR-PCT1 was cultured in a 100 mm φ culture dish (Falcon). After removing the culture solution and washing the cells with PBS, the cells were collected by a cell scraper (manufactured by IWAKI), and total RNA was extracted with an RNA extraction reagent (Trizol; manufactured by Gibco). Using reverse transcriptase (Rev Tra Ace: manufactured by TOYOBO), cDNA was synthesized from all the extracted RNAs, and expressed by PCR (exTacj; manufactured by Takara Shuzo). It was confirmed. Confirmation of the PCR amplification product was performed by electrophoresis on a 5% acrylamide gel. As a result, the cell line T R—PC T 1
3 1
おいてアンギオポェチン— 1 とォステオポェチン及び I C AM— 1 の発 現を確認した。 なお、 T R— P C T 2についても同様の結果が得られた。 3 1 In addition, the expression of angiopoietin-1 and osteopoetin and ICAM-1 was confirmed. Similar results were obtained for TR-PCT2.
4 - 5 (高密度培養によるマ トリ ツクス塊カルシウム沈着の確認） 上記実施例 4 一 1で得られた細胞株を単層培養し、 マトリ ックス塊に 沈着したカルシウムを V o n k o s s a染色にて確認した。 すなわち、 1 0 6個の細胞株 T R — P C T 1 を、 D M E Mにて 1 0 0 mm φ培養シ ャ一レ （Falcon社製） 及び 1 0 O mm c) I型コラーゲンコート培養シャ 一レ （IWAKI社製） に培養した。 Ι Ο Ο ιηπι φ Ι 型コラーゲンコート培 養シャーレの培養液には 1 O mMの 3—グリセ口リン酸を添加した。 3 日後培養液を除去し、 細胞を P B Sで洗浄した後、 8 mLの固定液 （ 0 . 1 %のダル夕一ルアルデヒ ドを含む P B S ) を加え、 室温で 1 5分放置 後、 蒸留水で 2回洗浄した。 8 mLの 5 %の硝酸銀水溶液 （Wako社製） を加え遮光し、 室温で 3 0分放置した後、 5 %の硝酸銀水溶液を除去し、 蒸留水で 2回洗浄した。 シャーレは室温で 3 0分露光させ、 観察は顕微 鏡で行った。 この結果、 細胞株 T R— P C T 1ではマ トリ ック塊にカル シゥムの沈着を認め、 コラーゲンコート培養シャーレではカルシウム沈 着の増加を確認した。 なお、 T R— P C T 2 についても同様の結果が得 られた。 4-5 (Confirmation of calcium deposit in matrix by high-density culture) The cell line obtained in Example 4-11 was cultured in a monolayer, and calcium deposited in the matrix was confirmed by Vonkossa staining. . That is, 1 0 6 cell lines TR - the PCT 1, (manufactured by Falcon) 1 0 0 mm phi cultured catcher Ichire in DMEM and 1 0 O mm c) I collagen coated culture Shah Ichire (IWAKI (Manufactured by the company). 1 OmM 3-phosphoric acid phosphate was added to the culture solution of the コ ー ト Ο ιιηπι φ Ι type collagen-coated culture dish. After 3 days, remove the culture solution, wash the cells with PBS, add 8 mL of a fixative (PBS containing 0.1% dal aldehyde), leave at room temperature for 15 minutes, and add distilled water. Washed twice. 8 mL of a 5% aqueous silver nitrate solution (manufactured by Wako) was added thereto, protected from light, allowed to stand at room temperature for 30 minutes, and then the 5% aqueous silver nitrate solution was removed and washed twice with distilled water. The petri dish was exposed at room temperature for 30 minutes, and observation was performed with a microscope. As a result, calcium deposits were observed in the matrix mass in the cell line TR-PCT1, and increased calcium deposition was observed in the collagen-coated culture dish. Similar results were obtained for TR-PCT2.
実施例 5 (血液脳関門再構築モデルの作製） Example 5 (Preparation of blood-brain barrier reconstruction model)
5 — 1 (共培養） 5 — 1 (co-culture)
上記実施例 2〜 4で調製した、 脳毛細血管内皮細胞株 （T R— B B B 1 3 ) とァス ト口サイ ト細胞株 （T R— A S T 9 3 2 ) との共培養 （以 下 「A +内皮細胞」 という）、 及び脳毛細血管内皮細胞株 （T R— B B B 1 3 ) とァス ト口サイ ト細胞株 （T R— A S T 9 3 2 ) と脳毛細血管周 皮細胞株 （T R— P C T 1 ) との共培養 （以下 「A + P +内皮細胞」 と いう） を行った。 なお、 脳毛細血管内皮細胞株 （T R— B B B 1 3 ) の Co-culture of the brain capillary endothelial cell line (TR-BBB 13) and the east mouth site cell line (TR-AST 932) prepared in Examples 2 to 4 above (hereinafter referred to as “A + Endothelial cells), cerebral capillary endothelial cell line (TR-BBB13), astral site cell line (TR-AST932), and cerebral capillary pericyte cell line (TR-PCT1). ) (Hereinafter referred to as “A + P + endothelial cells”). In addition, the brain capillary endothelial cell line (TR-BBBB13)
3 2
単独培養をコントロールとした。 3 2 A single culture was used as a control.
5 - 2 (使用細胞株の調製） 5-2 (Preparation of cell line used)
上記実施例 2により得られた T R _ B B B 1 3は、 D M E M 1 [D M E M (二ッスィ社製 ； # 0 5 9 1 5 ) に最終濃度で 1 0 %のゥシ胎児血 清 （ J R H社製； # 1 2 0 0 3 — 7 8 P)、 l O O UZm l のペニシリ ン、 1 0 0 /x g Zm l のス トレプトマイシン、 2 mMのグルタミン （G I B C O社製； # 1 0 3 7 8 — 0 1 6 )、 1 5 // g /m l の b E C G F (ベ一 リ ンガ一社製； # 1 0 3 3 4 8 4 )] に分散して、 コラーゲンタイプ I を コートした培養皿 （IWAKI社製 ； # 4 0 2 0 — 1 0 ) に播種し、 5 % C 02Z 9 5 % A I Rの条件下において 3 3 °Cで予め培養しておいた。 ま た、 上記実施例 3により得られた T R - A S T 9 3 2及び上記実施例 4 により得られた T R— P C T 1 は、 D M E M 2 [D M E M (二ッスィ社 製 ； # 0 5 9 1 5 ) に最終濃度で 1 0 %のゥシ胎児血清 （ J R H社製 ； # 1 2 0 0 3 — 7 8 P)、 l O O UZm l のペニシリン、 l O O g Zm 1 のス トレプトマイシン、 2 mMのグル夕ミン （ G I B C〇社製 ； # 1 0 3 7 8 — 0 1 6 )] が入った培養皿 （Corning社製 ； # 4 3 0 1 6 7 ) で、 5 % C 02 Z 9 5 % A I Rの条件下において 3 3 °Cで予め培養して おいた。 TR_BBB 13 obtained by the above Example 2 was added to DMEM 1 [DMEM (manufactured by Nissi; # 05915) # 105915] to give a final concentration of 10% fetal serum (manufactured by JRH; # 1203-78 P), 100 UZm1 penicillin, 100 / xg Zm1 streptomycin, 2 mM glutamine (GIBCO; # 10378-8) 16), 15 // g / ml of bECGF (manufactured by Behringer Inc .; # 103333484)], and a collagen type I-coated culture dish (manufactured by IWAKI) ; # 4 0 2 0 - 1 0) were seeded in advance cultured under conditions of 5% C 0 2 Z 9 5 % AIR at 3 3 ° C. Further, TR-AST932 obtained in Example 3 and TR-PCT1 obtained in Example 4 were converted to DMEM 2 [DMEM (Nissi; # 05915)]. 10% fetal serum at final concentration (manufactured by JRH; # 1203 — 78P), lOO UZml penicillin, lOOg Zm1 streptomycin, 2 mM glue evening Min (GIBC_〇 Ltd .; # 1 0 3 7 8 - 0 1 6)] that contains the culture dish; in (Corning Inc. # 4 3 0 1 6 7) , 5% C 0 2 Z 9 5% AIR The cells were pre-cultured at 33 ° C under the following conditions.
5 - 3 (非接触状態での培養） 5-3 (non-contact culture)
上記予め培養した細胞株を用いて、 図 1 に示すように、 非接触状態で の培養を行った。 D M E M 1 が入ったミ リ セル （ミ リ ポア社製 ； Using the previously cultured cell lines, non-contact culture was performed as shown in FIG. Millicell containing D M E M 1 (Millipore;
Millicell : 3 . 0 p. m c u l t u r e i n s e r t 、 3 0 mm d i a m e t e r 、 # P I T P 0 3 0 5 0 ) に上記 T R _ B B B 1 3 ( 1Millicell: 3.0 p.m c u l t u r e i n s e r t, 30 mm d i a m e t e r, # PIT P 0 3 0 5 0)
X I 0 5細胞 Zゥエル） を入れ、 上記 D M E M 2 の入った下側の 6 ゥェ リレプレートに上記 T R— A S T 9 3 2 ( 3 X 1 0 4細胞 Zゥエル）、 又はXI 0 5 cells Z Ueru) placed, the DMEM 2 of containing the lower 6 © E relay plate to the TR- AST 9 3 2 (3 X 1 0 4 cells Z Ueru), or
T R— A S T 9 3 2及び上記 T R— P C T 1 ( 1 X 1 0 5細胞 Zゥエル） TR- AST 9 3 2 and the TR- PCT 1 (1 X 1 0 5 cells Z Ueru)
3 3
を入れて、 5 % C 02Z 9 δ % A I Rの条件下において 3 3 °Cで 4日間 非接触状態で共培養した。 これらの非接触系培養における内皮細胞の酵 素活性の変化を、 血液脳関門の内皮細胞の酵素マーカーであるアル力リ フォスファターゼ（ A L P )ゃァーダル夕ミルトランスぺプチダーゼ（ Ύ - GT P) の活性を測定することにより調べた。 3 3 And co-cultured in a non-contact state at 33 ° C. for 4 days under the condition of 5% CO 2 Z 9 δ% AIR. Changes in the enzymatic activity of endothelial cells in these non-contact cultures were determined by the activity of the enzyme, an enzyme marker for endothelial cells at the blood-brain barrier, such as al-force phosphatase (ALP) ダ ル adal mil transpeptidase (Ύ-GTP). Was determined by measuring.
実施例 6 (酵素活性の測定） Example 6 (Measurement of enzyme activity)
6 - 1 (タンパク質濃度） 6-1 (protein concentration)
B C A酵素アツセィ試薬（BCAProteinAssay Reagent; PIRCE社製 ： # 5 3 1 — 2 0 7 2 1 ) の試薬 Aと試薬 Bを 5 0 : 1の割合で混合して 1 日間放置し、 ワーキングソリューショ ン （working solution) とした。 それぞれ 0. 1 m 1 の上記培養した 3種類のサンプル （コン トロール、 A +内皮細胞、 A+ P +内皮細胞） 及び B S A標準液に、 各 2 m l のヮ —キングソリューショ ンを加え、 3 7 °Cで 3 0分間インキュベートし、 室温まで冷却した後、 5 6 2 nmの波長における吸光度を測定した。 B S A標準液により作成した検量線によりの 3種類のサンプルのタンパク 質濃度をそれぞれ算出した。 Reagent A and reagent B of BCA enzyme assay reagent (BCAProtein Assay Reagent; manufactured by PIRCE: # 531-2072) were mixed at a ratio of 50: 1 and left for 1 day to prepare a working solution (working solution). solution). To 0.1 ml of each of the above three types of cultured samples (control, A + endothelial cells, A + P + endothelial cells) and BSA standard solution, add 2 ml of ヮ -King solution, and add After incubation with C for 30 minutes and cooling to room temperature, the absorbance at a wavelength of 562 nm was measured. The protein concentration of each of the three samples was calculated based on a calibration curve prepared using the BSA standard solution.
6 - 2 ( A L P活性の測定） 6-2 (ALP activity measurement)
次に、 AL Pテス トヮコー （^¥八 〇社製 ： # 2 7 4— 0 4 4 0 1 ) の基質緩衝液 0. 5 m l を 3 7 °Cで 3分間予めィンキュベーシヨ ンして おいたものに、 5 0 1 の上記培養した 3種類のサンプル （コントロー ル、 A +内皮細胞、 A + P十内皮細胞） を加えて 3 7 °Cで 1 5分間イン キュベ一シヨ ンし、 0. 0 2 Nの N a OH溶液を 5 m l加えた後よく振 り混ぜた。 これらの溶液を 4 0 5 nmの波長で吸光度を測定し、 予め作 成しておいた検量線から吸光度に相当する A L P活性値 （mUZm 1 ) を算出し、 上記蛋白濃度より 1 m gの蛋白質当たりの A L P活性値を求 めた。 結果を図 2に示す。 Next, 0.5 ml of a substrate buffer solution of ALP Test Co. (manufactured by Yappa Co., Ltd .: # 274-4041) was pre-incubated at 37 ° C for 3 minutes. Of the three cultured samples (control, A + endothelial cells, and A + P tenth endothelial cells) were added to the mixture, and the mixture was incubated at 37 ° C for 15 minutes to give 0.0 After adding 5 ml of 2N NaOH solution, the mixture was shaken well. The absorbance of these solutions was measured at a wavelength of 405 nm, and the ALP activity value (mUZm 1) corresponding to the absorbance was calculated from a calibration curve prepared in advance, and the protein concentration per 1 mg of protein was calculated from the above protein concentration. ALP activity value was determined. The result is shown in figure 2.
34
6 - 3 (ァー G T P活性の測定） 34 6-3 (A) Measurement of GTP activity
また、 ァー G T Pテス トヮコー （WAK〇社製 ： # 2 7 3 — 3 2 7 0 1 ) の基質緩衝液 3 m l を 3 7 °Cで 5分間予めィンキュベーシヨ ンして おいたものに、 1 5 1 の上記培養した 3種類のサンプル （コン トロー ル、 A +内皮細胞、 A十 P +内皮細胞） を加えて 3 7 °Cで正確に 3 0分 間インキュベーショ ンし、 4〜 5回振って混合させ、 発色液 Bを 1 0 0 II 1加えた後よく振り混ぜ、 室温にて 1 0分間放置した。 これらの溶液 を 5 5 0 nmの波長で吸光度を測定し、 予め作成しておいた検量線から 吸光度に相当するァー GT P活性値 （mUZm l ) を算出し、 上記蛋白 濃度より 1 m gの蛋白質当たりのァー G T P活性値を求めた。 結果を図 3に示す。 In addition, 3 ml of a substrate buffer solution of GTP Test Co., Ltd. (manufactured by WAK Corporation: # 273-32701) was pre-incubated at 37 ° C for 5 minutes. Add the three samples (Control, A + endothelial cells, A10P + endothelial cells) from 1 above, incubate at 37 ° C for exactly 30 minutes, and shake 4-5 times. After mixing, the coloring solution B was added to 100 II, shaken well, and allowed to stand at room temperature for 10 minutes. The absorbance of these solutions was measured at a wavelength of 550 nm, and the GTP activity value (mUZml) corresponding to the absorbance was calculated from a previously prepared calibration curve. The GTP activity value per protein was determined. The results are shown in Figure 3.
6 - 4 (測定結果の比較） 6-4 (Comparison of measurement results)
上記の結果から、 A L P活性値はそれぞれ、脳毛細血管内皮細胞株（T R— B B B 1 3 ) のみを培養したコントロールにおいては 3. 9 3 ± 0. 3 9 mUZmg、 ァス トロサイ ト細胞株 （T R— A S T 9 3 2 ) と共培 養した脳毛細血管内皮細胞株 （A +内皮細胞） においては 2 3. 5 ± 4. 6 9 mU/m g , 脳毛細血管周皮細胞株 （T R— P C T 1 ) とァス ト口 サイ 卜細胞株と共培養した脳毛細血管内皮細胞株 （Α + Ρ +内皮細胞） においては 2 5. 0 ± 1 . 6 mUZm gであった。 また、 ァ — GT P活 性値はそれぞれ、 コン トロールにおいては 4. 1 3 ± 0. 6 8 mU/m g、 A +内皮細胞においては 1 8. 5 ± 1. 6 mU/m g、 A+ P +内 皮細胞においては 1 0. 3 ± 0 · 5 mUZm gであった。 これらのこと から、 T R— B B B 1 3単独培養に比較して、 T R— A S T 9 3 2 と共 培養することで AL P活性が 6倍に増加し、 ァー GT P活性が 4. 5倍 に増加することがわかった。 また、 T R— P C T 1 と T R— A S T 9 3 2 と共培養した脳毛細血管内皮細胞株の A L P活性が 6倍に増加し、 Ύ From the above results, the ALP activity value was 3.93 ± 0.39 mUZmg in the control cultured only with the brain capillary endothelial cell line (TR-BBB13), and the Astrocyte cell line (TR — A brain capillary endothelial cell line (A + endothelial cell) co-cultured with AST 932) was 23.5 ± 4.69 mU / mg, and a brain capillary pericyte cell line (TR-PCT 1 ) And 25.0 ± 1.6 mUZmg in the brain capillary endothelial cell line (Α + Ρ + endothelial cells) co-cultured with the first mouth site cell line. The α-GTP activity values were 4.13 ± 0.68 mU / mg for control, 18.5 ± 1.6 mU / mg for A + endothelial cells, and A + P +, respectively. In endothelial cells, it was 10.3 ± 0.5 · 5 mUZmg. From these facts, the co-culture with TR-AST932 increased the ALP activity by a factor of 6 and increased the GTP activity by a factor of 4.5 compared to the TR-BBB13 alone culture. Was found to increase. In addition, ALP activity of the brain capillary endothelial cell line co-cultured with TR—PCT1 and TR—AST932 increased 6-fold.
35
一 GT P活性が 2. 5倍に増加することがわかった。 35 One GTP activity was found to increase 2.5-fold.
実施例 Ί (G L UT— 1の検出） Example Ί (Detection of G LUT-1)
7 - 1 (R T - P C R) 7-1 (R T-P C R)
上記脳毛細血管内皮細胞株のみを培養したもの （コン トロール） や、 ァス トロサイ ト細胞株と共培養した脳毛細血管内皮細胞株 （A +内皮細 胞） における G 3 P DH (ハウスキーピング遺伝子） や G L UT— 1 (グ ルコース トランスポー夕一 1 ) の発現を半定量的 RT— P C Rにより検 出した。 上記脳毛細血管内皮細胞株をミ リ セル （ミ リ ポア社製 ； Millicell： 3.0 u m culture insert、 30mm diameter、 # P I T P 0 3 0 5 0 ) で培養した。 培養液を除去し、 細胞を P B Sで洗浄した後、 セル スク レーパー （Iwaki 社製） にて細胞をかき集め、 R N A抽出試薬 (Trizol； Gibco社製） にて全 RN Aを抽出した。 逆転写酵素 （RevTra Ace： 東洋紡社製） を用いて、 抽出した全 RNA l gから c DNAを 合成した。 G3PDH (housekeeping gene) in the culture of the above-mentioned brain capillary endothelial cell line alone (control) and in the brain capillary endothelial cell line (A + endothelial cell) co-cultured with the astrocyte cell line ) And GLUT-1 (glucose transport 1) were detected by semi-quantitative RT-PCR. The above-mentioned brain capillary endothelial cell line was cultured in Millicell (Millipore; Millicell: 3.0 um culture insert, 30 mm diameter, # PITP350). After removing the culture medium and washing the cells with PBS, the cells were scraped with a cell scraper (manufactured by Iwaki), and all RNA was extracted with an RNA extraction reagent (Trizol; manufactured by Gibco). Using reverse transcriptase (RevTra Ace: Toyobo), cDNA was synthesized from 1 g of the extracted total RNA.
7 - 2 (プライマー） 7-2 (primer)
得られた c DNA ( 5 0 n gZ l ) 2. 0 1 に対して、 P C R反 応を行った。 P C R反応における各遺伝子のプライマーの組合せとして は、 G 3 P DH [G 3 P DH - F : 5 ' 一 A C C AC AGT C CAT G C CAT CAC— 3 ' (配列番号 3 )、 G 3 P DH - R : 5 ' — T C C A C CAC C C TGTT G C T GTA- 3 ' (配列番号 4)]、 G L UT— 1 [GL UT - 1 一 F ： 5 ' 一 GATGAT GAAC C TGTT G G C C T - 3 ' (配列番号 5 )、 GL UT— 1 — R : 5 ' 一 AG C GGAA C AG C T C CAAGATG - 3 ' (配列番号 6 )] をそれぞれ用いた。 7 - 3 ( P C R ) A PCR reaction was performed on the obtained cDNA (50 ngZl) 2.01. G3PDH [G3PDH-F: 5′-ACC AC AGT C CAT GC CAT CAC—3 ′ (SEQ ID NO: 3), G3PDH-R : 5 '— TCCAC CAC CC TGTT GCT GTA-3' (SEQ ID NO: 4)], GL UT-1 [GL UT-1 1 F: 5 'one GATGAT GAAC C TGTT GGCCT-3' (SEQ ID NO: 5), GL UT—1—R: 5 ′ 1 AG C GGAA C AG CTC CAAGATG-3 ′ (SEQ ID NO: 6)] was used, respectively. 7-3 (PCR)
上記 c D NA ( 5 0 n g,Zん i l ) 2 1 に、 D DWを 3 3. 8 1 、 1 0 X Ε X b u f f e r を 5. 0 1 、 2. 5 mMの d N T P s m To the above c D NA (50 ng, Z i n l) 21, set D DW to 33.8 1, 10 X Ε X b uf f er to 5.0 1, 2.5 mM dNTPSM.
36
i xを 4. 0 36 ix 4.0
の r E x T a qを 0. 2 1 、 2 の上記各プライマー を 2. 加え、 全量 5 0 z l で P C R反応を行った。 サーマルサイ クルのプログラムは、 最初のみ 94 °Cで 3分間変性させ、 その後 9 4°C で 1分間熱変性させ、 5 7 °Cで 1分間伸張させ、 7 2 で 1分間ァニー リングするというサイクルを 2 5回繰り返し、 最後に 7 2 °Cで 1 0分間 アニーリングを行った。その後、 P C R増幅産物をァガロースゲル（ 2 %) 電気泳動法により分離した後、 ェチジゥムブロミ ド染色を行い、 C C D イメージアナライザ一を用いて、 それぞれのバンドの強度を測定した。 結果を図 4に示す。 The rExTaq was added to each of the above-mentioned primers of 0.21 and 2, and a PCR reaction was carried out with a total amount of 50 zl. The thermal cycle program consists of a first denaturation at 94 ° C for 3 minutes, followed by heat denaturation at 94 ° C for 1 minute, extension at 57 ° C for 1 minute, and annealing at 72 for 1 minute. Was repeated 25 times, and finally annealing was performed at 72 ° C. for 10 minutes. Thereafter, the PCR amplification product was separated by agarose gel (2%) electrophoresis, and then stained with ethidium bromide, and the intensity of each band was measured using a CCD image analyzer. Fig. 4 shows the results.
7 - 4 (測定結果の比較） 7-4 (Comparison of measurement results)
上記結果から、 ァス トロサイ ト細胞株と共培養した脳毛細血管内皮細 胞株 （A +内皮細胞） の血液脳関門に発現するへキソース輸送担体の m RNAである G l u t 1 の発現量は、 G 3 P DHとの比較からして、 脳 毛細血管内皮細胞株を単独培養したもの （コントロール） に比べて 1 0 0倍増加していることがわかった。 産業上の利用可能性 From the above results, the expression level of Glut1, a mRNA for the hexose transporter expressed at the blood-brain barrier of the brain capillary endothelial cell line (A + endothelial cells) co-cultured with the astrocyte cell line, was In comparison with G3PDH, it was found that the number was increased 100-fold as compared with that obtained by culturing the brain capillary endothelial cell line alone (control). Industrial applicability
本発明によると、 よりインビボに近いインビト口の血液脳関門実験系 の再構築が可能となり、 従来の共培養と比べて条件的不死化細胞株を使 用した共培養系は飛躍的に細胞一細胞間相互作用が促進され、 血液脳関 門が本来の機能に近いところまで、 再現できることがわかった。 したが つて、 本発明の血液脳関門再構築モデル等を用いることにより、 血液脳 関門に関する基礎的研究成果が得られる上に、 薬物の血液脳透過機構に 基づいた中枢作用型薬物 （抗痴呆薬、 脳腫瘍治療薬、 ウィルス治療薬、 精神神経作用薬） や、 中枢での副作用が問題となる薬物のスクリーニン According to the present invention, it is possible to reconstruct an in vivo blood-brain barrier experiment system that is closer to in vivo, and a co-culture system using a conditional immortalized cell line is dramatically more cell-efficient than conventional co-culture. It has been shown that cell-cell interactions are promoted and the blood-brain barrier can be reproduced to a point close to its original function. Therefore, by using the blood-brain barrier reconstruction model and the like of the present invention, not only basic research results on the blood-brain barrier can be obtained, but also centrally acting drugs (anti-dementia drugs) based on the blood-brain permeation mechanism of drugs. , Brain tumor drugs, virus drugs, neuropsychiatric drugs) and drugs that cause central side effects
3 7
グを効率的に行うことができるだけでなく、 脳毛細血管内皮細胞に発現 する種々の受容体を標的とした薬物 （脳微小循環改善薬、 脳浮腫治療薬） の効果や脳毛細血管内皮細胞の障害 （脳血管障害性痴呆症ゃァ 3 7 In addition to being able to efficiently perform the treatment, the effects of drugs (cerebral microcirculation improving drugs, drugs for treating brain edema) targeting various receptors expressed on brain capillary endothelial cells, Disability (Cerebrovascular dementia)
マー型痴呆症） の評価が可能となる。 Evaluation can be performed.
3 8
3 8
Claims
1. ラッ ト由来の不死化脳毛細血管内皮細胞株と、 ラッ ト由来の不死化 ァス トロサイ ト細胞株とを共培養することを特徴とする血液脳関門再構 築モデルの作製方法。 1. A method for preparing a blood-brain barrier remodeling model, which comprises co-culturing a rat-derived immortalized brain capillary endothelial cell line and a rat-derived immortalized astrocyte cell line.
2. ラッ ト由来の不死化脳毛細血管内皮細胞株と、 ラッ ト由来の不死化 ァス トロサイ ト細胞株と、 ラッ ト由来の不死化脳毛細血管周皮細胞株と を共培養することを特徴とする血液脳関門再構築モデルの作製方法。 2. Co-culture of rat-derived immortalized brain capillary endothelial cell line, rat-derived immortalized astrocyte cell line, and rat-derived immortalized brain capillary pericyte cell line A method for producing a blood-brain barrier reconstruction model that is a feature.
3. ラッ ト由来の不死化脳毛細血管内皮細胞株が、 温度感受性変異株 S V 4 0 t s A 5 8のラージ T抗原遺伝子を導入したトランスジエニック ラッ ト由来の不死化脳毛細血管内皮細胞株であることを特徴とする請求 項 1又は 2記載の血液脳関門再構築モデルの作製方法。 3. An immortalized brain capillary endothelial cell line derived from a rat is a transgenic rat derived from a transgenic rat into which a large T antigen gene of a temperature-sensitive mutant SV40tsA58 has been introduced. 3. The method for producing a blood-brain barrier reconstruction model according to claim 1 or 2, wherein:
4. 温度感受性変異株 S V 4 0 t s A 5 8のラージ T抗原遺伝子を導入 したトランスジェニックラッ ト由来の不死化脳毛細血管内皮細胞株が、 TR— B B B 1 3 (F E PM Β Ρ— 6 8 7 3 ) であることを特徴とす る請求項 3記載の血液脳関門再構築モデルの作製方法。 4. An immortalized cerebral capillary endothelial cell line derived from a transgenic rat into which the large T antigen gene of the temperature-sensitive mutant SV40tsA58 was introduced was TR-BBB13 (FEPM-Β-68). 7. The method for producing a blood-brain barrier reconstruction model according to claim 3, wherein:
5. ラッ ト由来の不死化ァス トロサイ ト細胞株が、 温度感受性変異株 S ¥ 4 0 3 八 5 8のラージ丁抗原遺伝子を導入したトランスジエニック ラッ 卜由来の不死化ァス トロサイ ト細胞株であることを特徴とする請求 項 1又は 2記載の血液脳関門再構築モデルの作製方法。 5. An immortalized astrocyte cell line derived from a transgenic rat into which a rat-derived immortalized astrocyte cell line has been transfected with a temperature-sensitive mutant S ¥ 4,388,58 3. The method for producing a blood-brain barrier reconstruction model according to claim 1, wherein the strain is a strain.
6. 温度感受性変異株 S V 4 0 t s A 5 8のラージ T抗原遺伝子を導入 したトランスジエニックラッ ト由来の不死化ァス トロサイ ト細胞株が、 TR -A S T 9 3 2 (F E PM Β Ρ— 6 2 8 3 ) であることを特徴と する請求項 5記載の血液脳関門再構築モデルの作製方法。 6. An immortalized astrocyte cell line derived from a transgenic rat into which the large T antigen gene of the temperature-sensitive mutant SV40tsA58 was introduced was TR-AST932 (FE PM Β— 6. The method for producing a blood-brain barrier reconstruction model according to claim 5, characterized in that:
7. ラッ ト由来の不死化脳毛細血管周皮細胞株が、 温度感受性変異株 S V 4 0 t s A 5 8のラージ T抗原遺伝子を導入したトランスジエニック 7. Immortalized cerebral capillary pericyte cell line derived from a rat is transgenic in which the large T antigen gene of the temperature-sensitive mutant SV40tsA58 was introduced.
39
ラッ ト由来の不死化脳毛細血管周皮細胞株であることを特徴とする請求 項 2記載の血液脳関門再構築モデルの作製方法。 39 3. The method for producing a blood-brain barrier reconstruction model according to claim 2, which is an immortalized brain capillary pericyte cell line derived from a rat.
8 . 温度感受性変異株 S V 4 0 t s A 5 8のラージ T抗原遺伝子を導入 したトランスジエニックラッ ト由来の不死化脳毛細血管周皮細胞株が、 T R— P C T l ( F E R M Β Ρ— 7 0 2 4 ) であることを特徴とする 請求項 7記載の血液脳関門再構築モデルの作製方法。 8. An immortalized cerebral capillary pericyte cell line derived from a transgenic rat into which the large T antigen gene of the temperature-sensitive mutant SV40tsA58 was introduced was TR-PCTl (FERM -70). 24. The method for producing a blood-brain barrier reconstruction model according to claim 7, characterized in that:
9 . 共培養が、 不死化脳毛細血管内皮細胞株と不死化ァス トロサイ ト細 胞株とを非接触状態で、 又は不死化脳毛細血管内皮細胞株と不死化ァス トロサイ ト細胞株及び不死化脳毛細血管周皮細胞株とを非接触状態で培 養する、 非接触系共培養であることを特徴とする請求項 1〜 8のいずれ か記載の血液脳関門再構築モデルの作製方法。 9. The co-culture is performed without contacting the immortalized cerebral capillary endothelial cell line with the immortalized astrocytic cell line, or the immortalized cerebral capillary endothelial cell line and the immortalized astrocytic cell line; The method for producing a blood-brain barrier remodeling model according to any one of claims 1 to 8, wherein the method is a non-contact type co-culture in which the immortalized brain capillary pericyte cell line is cultured in a non-contact state. .
1 0 . 請求項 1〜 9のいずれか記載の血液脳関門再構築モデルを用いる スクリーニング方法であって、 共培養中又は共培養の前後に、 被検物質 を不死化脳毛細血管内皮細胞株と接触させ、 血液脳関門のマーカー遺伝 子の発現の程度を測定 · 評価することを特徴とする血液脳関門形成促進 又は抑制物質のスク リ一二ング方法。 10. A screening method using the blood-brain barrier reconstruction model according to any one of claims 1 to 9, wherein the test substance is immortalized during or before or after co-culture with a brain capillary endothelial cell line. A method for screening a substance that promotes or suppresses the formation of the blood-brain barrier, which comprises bringing into contact and measuring and evaluating the degree of expression of a marker gene of the blood-brain barrier.
1 1 . 請求項 1〜 9のいずれか記載の血液脳関門再構築モデルを用いる スクリーニング方法であって、 共培養中又は共培養の前後に、 被検物質 と血液脳関門透過物質又は血液脳関門非透過物質とを不死化脳毛細血管 内皮細胞株と接触させ、 これら血液脳関門透過物質又は血液脳関門非透 過物質の不死化脳毛細血管内皮細胞内への透過の程度を測定 · 評価する ことを特徴とする血液脳関門透過促進又は抑制物質のスクリ一二ング方 法。 11. A screening method using the blood-brain barrier reconstruction model according to any one of claims 1 to 9, wherein a test substance and a blood-brain barrier permeant or a blood-brain barrier are used during or before or after co-culture. The impermeable brain capillary endothelial cell line is brought into contact with an impermeable substance, and the degree of permeation of the blood brain barrier permeating substance or the blood brain barrier non-permeating substance into the immortalized brain capillary endothelial cells is measured and evaluated. A method for screening a substance that enhances or suppresses blood-brain barrier penetration.
1 2 . 請求項 1〜 9のいずれか記載の血液脳関門再構築モデルを用いる スクリーニング方法であって、 共培養中又は共培養の前後に、 被検物質 を不死化脳毛細血管内皮細胞株と接触させ、 不死化脳毛細血管内皮細胞 12. A screening method using the blood-brain barrier reconstruction model according to any one of claims 1 to 9, wherein the test substance is immortalized with the brain capillary endothelial cell line during or before or after co-culture. Contact and immortalize brain capillary endothelial cells
4 0
内への被検物質の透過の程度を測定 · 評価することを特徴とする血液脳 関門透過又は非透過物質のスク リーニング方法。 4 0 A method for screening a permeated or non-permeated substance at the blood-brain barrier, comprising measuring and evaluating the degree of penetration of a test substance into the inside.
1 3 . 請求項 1 0記載の血液脳関門形成促進又は抑制物質のスクリ一二 ング方法により得られる血液脳関門形成促進物質。 13. A blood-brain barrier formation promoting substance obtained by the method for screening a blood-brain barrier formation promoting or suppressing substance according to claim 10.
1 4 . 請求項 1 0記載の血液脳関門形成促進又は抑制物質のスク リー二 ング方法により得られる血液脳関門形成抑制物質。 14. A blood-brain barrier formation-inhibiting substance obtained by the method for screening a blood-brain barrier formation-promoting or suppressing substance according to claim 10.
1 5 . 請求項 1 1記載の血液脳関門透過促進又は抑制物質のスク リー二 ング方法により得られる血液脳関門透過促進物質。 15. A blood-brain barrier permeation enhancer obtained by the method for screening a blood-brain barrier permeation promoting or suppressing substance according to claim 11.
1 6 . 請求項 1 1記載の血液脳関門透過促進又は抑制物質のスク リー二 ング方法により得られる血液脳関門透過抑制物質。 16. A blood-brain barrier permeation inhibitor obtained by the method for screening a blood-brain barrier permeation promoter or inhibitor according to claim 11.
1 7 . 請求項 1 2記載の血液脳関門透過又は非透過物質のスク リーニン グ方法により得られる血液脳関門透過物質。 17. A blood-brain barrier permeable material obtained by the method for screening a blood-brain barrier permeable or non-permeable material according to claim 12.
1 8 . 請求項 1 2記載の血液脳関門透過又は非透過物質のスクリーニン グ方法により得られる血液脳関門非透過物質。 18. A blood-brain barrier impermeable substance obtained by the method for screening a blood-brain barrier permeated or impermeable substance according to claim 12.
1 9 . ラッ ト由来の不死化脳毛細血管内皮細胞株と、 ラッ ト由来の不死 化ァス ト口サイ ト細胞株とを共培養することにより得られることを特徴 とする血液脳関門のマーカー遺伝子の発現が増強した不死化脳毛細血管 内皮細胞株。 19. Blood-brain barrier marker obtained by co-culturing rat-immortalized brain capillary endothelial cell line and rat-immortalized fast mouth cell line Immortalized brain capillary endothelial cell line with enhanced gene expression.
2 0 . ラッ ト由来の不死化脳毛細血管内皮細胞株と、 ラッ ト由来の不死 化ァス トロサイ ト細胞株と、 ラッ ト由来の不死化脳毛細血管周皮細胞株 とを共培養することにより得られることを特徴とする血液脳関門のマー カー遺伝子の発現が増強した不死化脳毛細血管内皮細胞株。 20. Co-culturing a rat-derived immortalized brain capillary endothelial cell line, a rat-derived immortalized astrocyte cell line, and a rat-derived immortalized brain capillary pericyte cell line An immortalized brain capillary endothelial cell line having enhanced expression of a marker gene at the blood brain barrier, which is obtained by the following method.
2 1 . ラッ ト由来の不死化脳毛細血管内皮細胞株が、 温度感受性変異株 S V 4 0 t s A 5 8のラージ T抗原遺伝子を導入したトランスジェニッ クラッ 卜由来の不死化脳毛細血管内皮細胞株であることを特徴とする請 求項 1 9又は 2 0記載の血液脳関門のマーカー遺伝子の発現が増強した 21. An immortalized brain capillary endothelial cell line derived from a rat is a transgenic rat derived from a transgenic rat into which the large T antigen gene of the temperature-sensitive mutant SV40tsA58 has been introduced. Claims 19 or 20, wherein the expression of the marker gene for the blood-brain barrier is enhanced.
4 1
不死化脳毛細血管内皮細胞株。 4 1 Immortalized brain capillary endothelial cell line.
2 2. 温度感受性変異株 S V 4 0 t s A 5 8のラージ T抗原遺伝子を導 入したトランスジエニックラッ ト由来の不死化脳毛細血管内皮細胞株が. TR— B B B 1 3 (F E PM Β Ρ— 6 8 7 3 ) であることを特徴とす る請求項 2 1記載の血液脳関門のマーカー遺伝子の発現が増強した不死 化脳毛細血管内皮細胞株。 2 2. An immortalized brain capillary endothelial cell line derived from a transgenic rat into which the large T antigen gene of the temperature-sensitive mutant SV40tsA58 was introduced. TR—BBB1 3 (FE PM PM Β) 21. The immortalized brain capillary endothelial cell line according to claim 21, wherein the expression of a marker gene for the blood brain barrier is enhanced.
2 3. ラッ ト由来の不死化ァス トロサイ ト細胞株が、 温度感受性変異株 S V 4 0 t s A 5 8のラージ Τ抗原遺伝子を導入した トランスジェニッ クラッ ト由来の不死化ァス トロサイ ト細胞株であることを特徴とする請 求項 1 9又は 2 0記載の血液脳関門のマーカー遺伝子の発現が増強した 不死化脳毛細血管内皮細胞株。 2 3. The immortalized astrocyte cell line derived from the rat is a transgenic rat from which the large ク antigen gene of the temperature-sensitive mutant SV40tsA58 has been introduced. 21. The immortalized brain capillary endothelial cell line according to claim 19 or 20, wherein the expression of the marker gene for the blood brain barrier is enhanced.
2 4. 温度感受性変異株 S V 4 0 t s A 5 8のラージ T抗原遺伝子を導 入したトランスジエニックラッ ト由来の不死化ァス ト口サイ ト細胞株が T R -A S T 9 3 2 (F E PM Β Ρ _ 6 2 8 3 ) であることを特徴と する請求項 2 3記載の血液脳関門のマーカ一遺伝子の発現が増強した不 死化脳毛細血管内皮細胞株。 2 4. An immortalized fast mouth cell line derived from a transgenic rat into which the large T antigen gene of the temperature-sensitive mutant SV40tsA58 was introduced was TR-AST932 (FEPM The immortalized brain capillary endothelial cell line according to claim 23, wherein the expression of the marker gene for the blood brain barrier is enhanced.
2 5. ラッ ト由来の不死化脳毛細血管周皮細胞株が、 温度感受性変異株 S V 4 0 t s A 5 8のラージ Τ抗原遺伝子を導入した トランスジェニッ クラッ 卜由来の不死化脳毛細血管周皮細胞株であることを特徴とする請 求項 2 0記載の血液脳関門のマーカー遺伝子の発現が増強した不死化脳 毛細血管内皮細胞株。 2 5. Immortalized cerebral capillaries derived from the rat were transformed with the immortalized cerebral capillaries derived from a transgenic rat into which the large Τ antigen gene of the temperature-sensitive mutant SV40tsA58 was introduced. The immortalized brain capillary endothelial cell line according to claim 20, wherein the expression of a marker gene for the blood-brain barrier is enhanced.
2 6. 温度感受性変異株 S V 4 0 t s A 5 8のラージ T抗原遺伝子を導 入したトランスジェニックラッ ト由来の不死化脳毛細血管周皮細胞株が. T R— P C T 1 (F E RM B P— 7 0 2 4 ) であることを特徴とする 請求項 2 5記載の血液脳関門のマーカー遺伝子の発現が増強した不死化 脳毛細血管内皮細胞株。 2 6. An immortalized brain capillary pericyte cell line derived from a transgenic rat into which the large T antigen gene of the temperature-sensitive mutant SV40tsA58 was introduced. TR—PCT1 (FE RM BP—7 An immortalized brain capillary endothelial cell line having enhanced expression of a marker gene for the blood-brain barrier according to claim 25, which is characterized in that:
42
42
2 7 . 共培養が、 不死化脳毛細血管内皮細胞株と不死化ァス トロサイ ト 細胞株とを非接触状態で、 又は不死化脳毛細血管内皮細胞株と不死化ァ ス トロサイ 卜細胞株及び不死化脳毛細血管周皮細胞株とを非接触状態で 培養する、 非接触系共培養であることを特徴とする請求項 1 9〜 2 6の いずれか記載の血液脳関門のマーカー遺伝子の発現が増強した不死化脳 毛細血管内皮細胞株。 27. The co-culture, in which the immortalized brain capillary endothelial cell line and the immortalized astrocyte cell line are not in contact with each other, or the immortalized brain capillary endothelial cell line and the immortalized astrocyte cell line, The expression of the marker gene of the blood-brain barrier according to any one of claims 19 to 26, wherein the immortalized brain capillary pericyte cell line is cultured in a non-contact state, which is a non-contact type co-culture. Improved immortalized brain capillary endothelial cell line.
2 8 . 血液脳関門のマーカー遺伝子が、 アルカリフォスファターゼ遺伝 子、 ァ ーダルタミールトランスぺプチダーゼ遺伝子、 G 1 u t 1遺伝子 から選ばれる 1種又は 2種以上の遺伝子であることを特徴とする請求項 1 9〜 2 7のいずれか記載の血液脳関門のマーカー遺伝子の発現が増強 した不死化脳毛細血管内皮細胞株。 28. The blood brain barrier marker gene is one or more genes selected from alkaline phosphatase gene, adartamyl transpeptidase gene, and G1ut1 gene. An immortalized brain capillary endothelial cell line, wherein the expression of the blood-brain barrier marker gene according to any one of 19 to 27 is enhanced.
2 9 . アルカリフォスファタ一ゼ遺伝子の発現が、 共培養することなく 単独培養したときと比べて 6倍以上増強したことを特徴とする請求項 2 8記載の血液脳関門のマーカー遺伝子の発現が増強した不死化脳毛細血 管内皮細胞株。 29. The expression of the marker gene for the blood-brain barrier according to claim 28, wherein the expression of the alkaline phosphatase gene is enhanced by 6 times or more as compared with the case of single culture without co-culture. Enhanced immortalized brain capillary endothelial cell line.
3 0 . 7 "—ダルタミールトランスべプチダーゼ遺伝子の発現が、 共培養 することなく単独培養したときと比べて 2倍以上増強したことを特徴と する請求項 2 8又は 2 9記載の血液脳関門のマーカー遺伝子の発現が増 強した不死化脳毛細血管内皮細胞株。 30. The blood-brain barrier according to claim 28 or 29, wherein the expression of the 30.7 "-daltamyl transbeptidase gene is enhanced by a factor of 2 or more as compared to the case of single culture without co-culture. An immortalized brain capillary endothelial cell line in which expression of a marker gene has been enhanced.
3 1 . G 1 u t 1遺伝子の発現が、 共培養することなく単独培養したと きと比べて 1 0 0倍以上増強したことを特徴とする請求項 2 8〜 3 0の いずれか記載の血液脳関門のマーカー遺伝子の発現が増強した不死化脳 毛細血管内皮細胞株。 31. The blood according to any one of claims 28 to 30, wherein the expression of the G1ut1 gene has been enhanced 100 times or more as compared with a single culture without co-culture. Immortalized brain capillary endothelial cell line with enhanced expression of brain barrier marker genes.
3 2 . 被検物質と、 請求項 1 9〜 3 1 のいずれか記載の血液脳関門のマ 一力一遺伝子の発現が増強した不死化脳毛細血管内皮細胞株とを接触さ せ、 血液脳関門のマーカー遺伝子の発現増強の程度を測定 · 評価するこ 32. Contacting a test substance with an immortalized brain capillary endothelial cell line having enhanced expression of the blood-brain barrier ma-chi gene in any one of claims 19 to 31, Measure and evaluate the degree of enhancement of the expression of barrier marker genes.
4 3
とを特徴とする血液脳関門形成促進又は抑制物質のスク リーニング方法,4 3 A method for screening a substance promoting or inhibiting blood-brain barrier formation,
3 3 . 被検物質と血液脳関門透過物質又は血液脳関門非透過物質と、 請 求項 1 9〜 3 1のいずれか記載の血液脳関門のマーカー遺伝子の発現が 増強した不死化脳毛細血管内皮細胞株とを接触させ、 これら血液脳関門 透過物質又は血液脳関門非透過物質の該不死化脳毛細血管内皮細胞内へ の透過の程度を測定 · 評価することを特徴とする血液脳関門透過促進又 は抑制物質のスクリーニング方法。 33. A test substance, a blood-brain barrier permeable substance or a blood-brain barrier impervious substance, and an immortalized brain capillary with enhanced expression of the blood-brain barrier marker gene according to any one of claims 19 to 31. An endothelial cell line, and measuring and evaluating the degree of penetration of these blood-brain barrier permeable substances or blood-brain barrier non-permeable substances into the immortalized brain capillary endothelial cells. A screening method for promoting or inhibiting substances.
3 4 . 被検物質と、 請求項 1 9〜 3 1 のいずれか記載の血液脳関門のマ 一力一遺伝子の発現が増強した不死化脳毛細血管内皮細胞株とを接触さ せ、 該不死化脳毛細血管内皮細胞内への被検物質の透過の程度を測定 - 評価することを特徴とする血液脳関門透過又は非透過物質のスクリー二 ング方法。 34. Contacting the test substance with an immortalized brain capillary endothelial cell line having enhanced expression of the blood-brain-barrier ma-chimone gene according to any one of claims 19 to 31, wherein the immortalization is performed. A method for screening a permeated or non-permeated substance of the blood-brain barrier, which comprises measuring and evaluating the degree of penetration of a test substance into keratinized capillary endothelial cells.
3 5 . 請求項 3 2記載の血液脳関門形成促進又は抑制物質のスクリー二 ング方法により得られる血液脳関門形成促進物質。 35. A blood-brain barrier formation promoting substance obtained by the method for screening a blood-brain barrier formation promoting or inhibiting substance according to claim 32.
3 6 . 請求項 3 2記載の血液脳関門形成促進又は抑制物質のスクリー二 ング方法により得られる血液脳関門形成抑制物質。 36. A blood-brain barrier formation-inhibiting substance obtained by the method for screening a blood-brain barrier formation-promoting or suppressing substance according to claim 32.
3 7 . 請求項 3 3記載の血液脳関門透過促進又は抑制物質のスクリー二 ング方法により得られる血液脳関門透過促進物質。 37. A blood-brain barrier permeation-enhancing substance obtained by the method for screening a blood-brain barrier permeation-promoting or inhibiting substance according to claim 33.
3 8 . 請求項 3 3記載の血液脳関門透過促進又は抑制物質のスクリー二 ング方法により得られる血液脳関門透過抑制物質。 38. A blood-brain barrier permeation-inhibiting substance obtained by the method for screening a substance for promoting or inhibiting blood-brain barrier permeation according to claim 33.
3 9 . 請求項 3 4記載の血液脳関門透過又は非透過物質のスクリーニン グ方法により得られる血液脳関門透過物質。 39. A blood-brain barrier permeable substance obtained by the method for screening a blood-brain barrier permeable or non-permeable substance according to claim 34.
4 0 . 請求項 3 4記載の血液脳関門透過又は非透過物質のスクリーニン グ方法により得られる血液脳関門非透過物質。 40. A blood-brain barrier impermeable substance obtained by the method for screening a blood-brain barrier permeated or impermeable substance according to claim 34.
4 4
4 4
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| US20220010258A1 (en) * | 2017-08-10 | 2022-01-13 | Universite De Poitiers | Device that can serve as a hemato-encephalitic barrier model |
| WO2021113512A1 (en) | 2019-12-04 | 2021-06-10 | The Board Of Trustees Of The Leland Stanford Junior University | Enhancing blood-brain barrier drug transport by targeting endogenous regulators |
| EP4069368A4 (en) * | 2019-12-04 | 2023-08-09 | The Board of Trustees of the Leland Stanford Junior University | IMPROVING BLOOD-BRAIN BARRIER DRUG TRANSPORT BY TARGETING ENDOGENIC REGULATORS |
| EP4431524A3 (en) * | 2019-12-04 | 2024-12-18 | The Board Of Trustees Of The Leland Stanford Junior University | Enhancing blood-brain barrier drug transport by targeting endogenous regulators |
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| JP2001238681A (en) | 2001-09-04 |
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