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WO2001064849A1 - Modele reconstituant la barriere hemato-encephalique obtenu par co-culture - Google Patents

Modele reconstituant la barriere hemato-encephalique obtenu par co-culture Download PDF

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Publication number
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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PCT/JP2001/001017
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Japanese (ja)
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Tetsuya Terasaki
Emi Nakashima
Hisashi Iizasa
Ken-Ichi Hosoya
Kenji Hattori
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Japan Science And Technology Corporation
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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

L'invention porte sur un modèle reconstituant la barrière hémato-encéphalique, très utile pour des études fondamentales sur le mécanisme de perméation de ladite barrière et le criblage: de médicaments dont l'activité principale est liée audit mécanisme, de médicaments présentant des effets secondaires sur le centre, de médicaments ciblant différents récepteurs exprimés dans les cellules endothéliales des capillaires du cerveau, ou de médicaments ciblant des troubles dans les cellules endothéliales des capillaires du cerveau. Ledit modèle est obtenu par co-culture d'une lignée de cellules endothéliales immortalisées des capillaires du cerveau provenant d'un rat transgénique présentant de gros gènes d'antigène T de mutants thermosensibles SV 40 tsA58 et d'une lignée d'astrocytes immortalisés provenant du même rat et à l'état de non contact, ou bien par co-culture des deux susdites lignées avec de plus une lignée de cellules adventives immortalisées des capillaires provenant du même rat et à l'état de non contact.
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WO2003025206A1 (fr) * 2001-09-19 2003-03-27 The Cleveland Clinic Foundation Dispositif et appareil de modelisation de culture cellulaire et tissulaire, et mode d'utilisation
CN102220281A (zh) * 2011-04-19 2011-10-19 陕西省食品药品检验所 一种肝细胞与Kupffer细胞共培养体系及其应用
US10143187B2 (en) 2017-02-17 2018-12-04 Denali Therapeutics Inc. Transferrin receptor transgenic models
WO2019030380A1 (fr) * 2017-08-10 2019-02-14 Universite De Poitiers Dispositif pouvant servir de modèle de barrière hémato-encéphalique
US10457717B2 (en) 2017-02-17 2019-10-29 Denali Therapeutics Inc. Engineered polypeptides
WO2021113512A1 (fr) 2019-12-04 2021-06-10 The Board Of Trustees Of The Leland Stanford Junior University Amélioration du transport de médicament à travers la barrière hémato-encéphalique par ciblage de régulateurs endogènes
US11795232B2 (en) 2017-02-17 2023-10-24 Denali Therapeutics Inc. Engineered transferrin receptor binding polypeptides

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JP2011200224A (ja) * 2010-03-03 2011-10-13 Nikko Chemical Co Ltd 経皮吸収評価装置及び評価方法
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SG11201803143YA (en) * 2015-10-19 2018-05-30 Emulate Inc Microfluidic model of the blood brain barrier
JP6831119B2 (ja) * 2016-04-15 2021-02-17 国立大学法人山口大学 血液脳関門インヴィトロモデルおよび血液脳関門インヴィトロモデルの作製方法
WO2018052948A1 (fr) 2016-09-13 2018-03-22 Angiocrine Bioscience, Inc. Barrière hémato-encéphalique comprenant des cellules endothéliales modifiées
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JP7277874B2 (ja) * 2019-03-27 2023-05-19 国立大学法人大阪大学 脳血管モデル及びデバイス
JP7336154B2 (ja) * 2019-10-18 2023-08-31 国立研究開発法人農業・食品産業技術総合研究機構 血液組織関門インビトロモデル、及び薬物の血液組織関門移行性評価方法
JPWO2021149679A1 (fr) * 2020-01-20 2021-07-29

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WO2003025206A1 (fr) * 2001-09-19 2003-03-27 The Cleveland Clinic Foundation Dispositif et appareil de modelisation de culture cellulaire et tissulaire, et mode d'utilisation
US6667172B2 (en) * 2001-09-19 2003-12-23 The Cleveland Clinic Foundation Cell and tissue culture modeling device and apparatus and method of using same
CN102220281A (zh) * 2011-04-19 2011-10-19 陕西省食品药品检验所 一种肝细胞与Kupffer细胞共培养体系及其应用
CN102220281B (zh) * 2011-04-19 2013-01-30 陕西省食品药品检验所 一种肝细胞与Kupffer细胞共培养体系及其应用
US11732023B2 (en) 2017-02-17 2023-08-22 Denali Therapeutics Inc. Engineered polypeptides
US10457717B2 (en) 2017-02-17 2019-10-29 Denali Therapeutics Inc. Engineered polypeptides
US11612150B2 (en) 2017-02-17 2023-03-28 Denali Therapeutics Inc. Transferrin receptor transgenic models
US10143187B2 (en) 2017-02-17 2018-12-04 Denali Therapeutics Inc. Transferrin receptor transgenic models
US11795232B2 (en) 2017-02-17 2023-10-24 Denali Therapeutics Inc. Engineered transferrin receptor binding polypeptides
US11912778B2 (en) 2017-02-17 2024-02-27 Denali Therapeutics Inc. Methods of engineering transferrin receptor binding polypeptides
US12162948B2 (en) 2017-02-17 2024-12-10 Denali Therapeutics Inc. Methods of engineering transferrin receptor binding polypeptides
WO2019030380A1 (fr) * 2017-08-10 2019-02-14 Universite De Poitiers Dispositif pouvant servir de modèle de barrière hémato-encéphalique
FR3070045A1 (fr) * 2017-08-10 2019-02-15 Universite De Poitiers Dispositif pouvant servir de modele de barriere hemato-encephalique
US20220010258A1 (en) * 2017-08-10 2022-01-13 Universite De Poitiers Device that can serve as a hemato-encephalitic barrier model
WO2021113512A1 (fr) 2019-12-04 2021-06-10 The Board Of Trustees Of The Leland Stanford Junior University Amélioration du transport de médicament à travers la barrière hémato-encéphalique par ciblage de régulateurs endogènes
EP4069368A4 (fr) * 2019-12-04 2023-08-09 The Board of Trustees of the Leland Stanford Junior University Amélioration du transport de médicament à travers la barrière hémato-encéphalique par ciblage de régulateurs endogènes
EP4431524A3 (fr) * 2019-12-04 2024-12-18 The Board Of Trustees Of The Leland Stanford Junior University Amélioration du transport de médicament de barrière hémato-encéphalique par ciblage de régulateurs endogènes

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