+

WO2006112400A1 - Procede d'examen de la propriete metastatique et de la capacite d'infiltration du cancer - Google Patents

Procede d'examen de la propriete metastatique et de la capacite d'infiltration du cancer Download PDF

Info

Publication number
WO2006112400A1
WO2006112400A1 PCT/JP2006/307949 JP2006307949W WO2006112400A1 WO 2006112400 A1 WO2006112400 A1 WO 2006112400A1 JP 2006307949 W JP2006307949 W JP 2006307949W WO 2006112400 A1 WO2006112400 A1 WO 2006112400A1
Authority
WO
WIPO (PCT)
Prior art keywords
gpr48
cancer
expression
protein
cell
Prior art date
Application number
PCT/JP2006/307949
Other languages
English (en)
Japanese (ja)
Inventor
Masatoshi Kitagawa
Yun Gao
Kyoko Kitagawa
Original Assignee
National University Corporation Hamamatsu University School Of Medicine
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by National University Corporation Hamamatsu University School Of Medicine filed Critical National University Corporation Hamamatsu University School Of Medicine
Priority to JP2007526861A priority Critical patent/JP4887505B2/ja
Publication of WO2006112400A1 publication Critical patent/WO2006112400A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH

Definitions

  • the present invention relates to a method for examining cancer, particularly cancer with a poor prognosis, and more particularly, a gene involved in metastatic invasive ability in cells and tissues of a patient suspected of having cancer, particularly with a poor prognosis.
  • the present invention relates to a method for examining metastasis and invasiveness of cancer by examining the expression of. Background art
  • GPR48 which was found by the present inventors to be associated with cancer with poor prognosis and metastasis invasion of cancer cells, is a G protein binding containing a leucine-rich repeat belonging to a superfamily of proteins having 7-pass domains. Force known to be a receptor The function was unknown (Non-Patent Documents 4 and 5).
  • Non-Patent Document 1 Nature Medicine 1997 Feb; 3 (2): 222-5.
  • Non-Patent Document 2 Nature Medicine 1997 Feb; 3 (2): 227_30.
  • Non Patent Literature 3 Nature Medicine 1997 Feb; 3 (2): 231_4
  • Non-Patent Document 4 Mol Endocrinol. 2004 Sep; 18 (9): 2241-54. Epub 2004 Jun 10.
  • Non-Patent Document 5 Mol Endocrinol. 1998 Dec; 12 (12): 1830-45.
  • Non-Patent Documents 1 to 4 To elucidate the cause, we analyzed the gene expression caused by the decreased expression of p27 Kipl , and elucidated the cause of the poor prognosis.
  • the present invention provides a method for screening cancer, particularly cancer with poor prognosis.
  • the present inventors created a model in which the expression level of p27 Kipl was reduced, and analyzed the fluctuating gene using a microarray. That is, a cell (HCT-hp27-hKO) in which the p27 Kipl gene of the human colorectal cancer cell line HCT116 was knocked out by gene targeting method was created, and it was confirmed that the expression level of p27 Kipl was reduced. did. Genes whose expression was changed in HCT-hp27-hKO compared to the parent strain HCT116 were analyzed using a microarray. As a result, it was revealed that the expression of the G protein-coupled receptor GPR48 gene (AF25 7182) was significantly increased in HCT-hp27-hKO.
  • GPR48 has a function of enhancing the invasion ability of cancer cells, and have completed the present invention.
  • the present invention is a method for detecting cancer metastasis comprising detecting the expression of G protein-coupled receptor GPR48 in a cell or tissue to be examined. Is a method for examining the invasion ability of cancer, comprising detecting the expression of GPR48 in the tissue.
  • the present invention uses GRT48 expression levels in cancer tissue samples of cancer patients by quantitative RT-PCR or immunohistochemical methods such as immunohistochemical staining using enzyme-immunoantibody methods such as anti-GPR48 antibodies. It is a method of diagnosing the possibility.
  • the present invention detects the expression of GPR48 based on the finding that the G protein-coupled receptor GPR48 has a function of promoting the invasive ability of cancer cells and serves as an indicator of cancer invasive metastasis. This is a screening method for metastasis and invasion ability of cancer.
  • the cancer to be examined may be any cancer, for example, colon cancer, breast cancer, stomach cancer, lung cancer, biliary tract cancer (bile duct cancer, gallbladder cancer), knee cancer, esophageal cancer, hepatocellular carcinoma, laryngeal cancer, pharyngeal cancer. , Thyroid cancer, uterine cancer, ovarian cancer, renal cell cancer, prostate cancer, bladder cancer, malignant melanoma, brain tumor, etc. It is done.
  • the cell or tissue to be examined is also a biopsy sample from a suspected cancer patient or a patient suspected of cancer by a surgical technique, preferably a cell or tissue collected from a subject. More preferably, it is a cell or tissue.
  • the method for detecting the expression of GPR48 is not particularly limited, but includes antigen-antibody reaction, RT-PCR, cDNA microarray, Northern blotting, mass spectrometry analysis, protein chip analysis, and the like. These may be performed according to conventional methods in each field.
  • RT-PCR may be normal RT-PCR or real-time RT-PCR.
  • Primers that specifically amplify human GPR48 mRNA are designed from human GPR48 (SEQ ID NO: 1) according to conventional methods.
  • the primer sequence is not particularly limited as long as it is a sequence in human GPR48 mRNA and exhibits specific amplification.
  • the expression level of GPR48 in the cells can be examined by RT-PCR using two appropriate primers in the sequence of G protein-coupled receptor GPR48.
  • an antibody using a peptide corresponding to the extracellular domain of human GPR48 (SEQ ID NO: 2) as an antigen.
  • This extracellular domain is amino acids Nol to 542 of human GPR48 (SEQ ID NO: 2), and may be an antibody against the entire extracellular domain or a part thereof.
  • the antibody may be a polyclonal antibody or a monoclonal antibody.
  • the antibody of the present invention reacts with GPR48 present in a specimen. Further, a probe that recognizes this antibody is reacted. Examples of this probe include anti-human IgG antibody, protein G, protein A, and protein L. This probe is usually labeled. These labels include radioisotopes ( 125 ⁇ ), enzymes (peroxidase)
  • a DNA chip on which a number of probes (DNA) corresponding to mRNA derived from human GPR48 (SEQ ID NO: 1) are immobilized is prepared.
  • a cell fraction or tissue force to be examined or RNA fraction or mRNA is prepared, and DNA complementary thereto is labeled with a fluorescent dye and synthesized.
  • the expression of GPR48 can be detected by measuring the fluorescence by pairing this with the above DNA chip.
  • RNA fractionated by electrophoresis is immobilized on a nitrocellulose membrane or the like from a gel. This can be paired with a labeled DNA (probe) prepared from human GPR48 (SEQ ID NO: 1), and the expressed mRNA can be detected to know the expression of GPR48.
  • protein expression can be extracted from a sample, analyzed with a mass spectrometer, and the amount of fragments having the amino acid sequence of GPR48 can be measured to determine the expression of GPR48.
  • Isogen manufactured by Wako
  • RNA was converted into a vertical form, and then used for hundamorikovima 1 ⁇ ; cDNA was synthesized with supenscript Reverse ranscnptase (Invitrogen).
  • Primer set and sample cDNA are stored in a reaction solution containing thermostable polymerase. Set on a PCR machine (Light Cycler, Roche), 94. The amount of GPR48 mRNA was quantified by performing cyclic amplification at 54 ° C. for 20 seconds and 72 ° C. for 20 seconds after heat denaturation for C 15 seconds.
  • Matrigel invasion chamber (Matrigel invasion chamber, manufactured by BD Bioscience, 24-wel 1 plate size), Matrigel coated PET membrane (PET membrane, pore size 8.0- ⁇ m) chamber These cells were seeded at 5 ⁇ 10 4 cells / well.
  • remove from the 37 ° C CO incubator remove cells above chamber 1 with a cotton swab, pass through Matrigenore, and infiltrate cells below chamber 1 with Diff-Quik kit (International Reagents Co ⁇ ). oration) and observed with a microscope at 100 times magnification, and the cells in all fields were counted.
  • GPR48 expression plasmid pcDNA4-HisMax_hGPR48
  • empty vector pcDNA4_HisMax
  • Figure 2 is a photograph of stained infiltrated cells.
  • Figure 3 shows the results in a graph and statistical processing. * Indicates that GPR48-19 and GPR48-33 are significantly more invasive than control vectors at a risk rate of 0.01 or less. As a result, it was found that GPR48-19 and GPR48-33 cells expressing GPR48 mRNA at a high level were clearly more invasive than the control group Vector 2 and Vector 6.
  • GPR48 can promote cancer cell invasion.
  • GPR48 expression was analyzed by quantitative RT-PCR in 29 human colorectal cancer specimens with informed consent.
  • Figure 4 shows the expression level of GPR 48 mRNA in 29 cancerous and normal areas.
  • a peptide (CQEQKMLRTLDL) having a sequence of amino acids 339 to 350 at the extracellular position of human GPR48 (SEQ ID NO: 2) was chemically synthesized, conjugated with KLH, and then immunized with a rabbit. After confirming that the antibody titer had increased, whole blood was collected to obtain antiserum.
  • Extracts of 293 cells or HCT116 cells transfected with a GPR48 expression vector (pcDNA4-HisMax-hGPR48) were separated by SDS polyacrylamide electrophoresis and subjected to Western plot analysis. As a result, a human GPR48 band was detected at a molecular weight of 104 kD.
  • Immunohistological staining was performed using the Histofme SAB kit (Nichirei). Specifically, paraffin-embedded tissue sections were deparaffinized and then treated with a microwave oven in 10 mM citrate buffer to remove endogenous peroxidase with 0.3% hydrogen peroxide. Next, it was immersed in a purified anti-GPR48 antibody diluted 1: 200 and allowed to react overnight at 4 ° C. Subsequently, it was reacted with a piotine-conjugated secondary antibody (anti-rabbit IgG), then reacted with streptavidin-conjugated horseradish peroxidase, and after washing, it was immersed in a coloring solution containing diaminobenzidine for color development. Furthermore, the nucleus was dyed blue with hematoxylin, mounted with cover glass, and observed with a microscope.
  • FIG. 5 shows a staining example in which the same colon cancer specimen as in Example 1 was analyzed using this immunohistological staining method.
  • FIG. 1 is a graph showing the expression level of GPR48 mRNA in human colon cancer cells into which a GPR48 expression plasmid has been introduced.
  • FIG. 3 A graph showing the number of cells infiltrated in human colon cancer cells into which a GPR48 expression plasmid was introduced.
  • the vertical axis shows the number of cells that infiltrated and passed through the pores (holes) of a Matrigel-coated PET membrane (pore size 8 ⁇ 0 ⁇ m) (total number of infiltrated cells per chamber). Number).
  • FIG. 5 is a diagram showing expression analysis (immunohistological staining) of GPR48 in human colon cancer specimens.
  • black circles blue in color, and due to hematoxylin-eosin staining
  • GPR48 the part of the cytoplasm around the nucleus that is stained gray (gray near black, dark brown in color)
  • GPR48 the part of the cytoplasm around the nucleus that is stained gray (gray near black, dark brown in color)
  • the unstained part (white) in the meantime is stromal cells (normal).
  • the response of GPR48 shown in gray is very weak.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Zoology (AREA)
  • Physics & Mathematics (AREA)
  • Urology & Nephrology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Oncology (AREA)
  • Food Science & Technology (AREA)
  • Endocrinology (AREA)
  • Hospice & Palliative Care (AREA)
  • General Physics & Mathematics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

[PROBLEMES] Apporter un procédé d'examen du cancer à faible pronostic. [RESOLUTION DE PROBLEMES] Il a été découvert que l'expression d'un gène GPR48 du récepteur couplé aux protéines G (AF257182) est considérablement accélérée en cas de cancer à faible pronostic. Les résultats des études ultérieures visant à clarifier le rapport entre GPR48 et la métastase ont indiqué et que le GPR48 a une fonction d'accélération de la capacité d'infiltration des cellules cancéreuse et les résultats obtenus sont appliqués au diagnostic du cancer à faible pronostic. A cet effet, on met en oeuvre un procédé d'examen de la propriété métastatique du cancer et un procédé d'examen de la capacité d'infiltration du cancer consistant à détecter l'expression du récepteur GPR48 couplé aux protéines G dans une cellule ou un tissu à examiner.
PCT/JP2006/307949 2005-04-18 2006-04-14 Procede d'examen de la propriete metastatique et de la capacite d'infiltration du cancer WO2006112400A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2007526861A JP4887505B2 (ja) 2005-04-18 2006-04-14 癌の転移性及び浸潤能の検査方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2005119289 2005-04-18
JP2005-119289 2005-04-18

Publications (1)

Publication Number Publication Date
WO2006112400A1 true WO2006112400A1 (fr) 2006-10-26

Family

ID=37115118

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2006/307949 WO2006112400A1 (fr) 2005-04-18 2006-04-14 Procede d'examen de la propriete metastatique et de la capacite d'infiltration du cancer

Country Status (2)

Country Link
JP (1) JP4887505B2 (fr)
WO (1) WO2006112400A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009100737A (ja) * 2007-10-01 2009-05-14 Japan Health Science Foundation α−アクチニン−４遺伝子のコピー数または発現レベルを指標とした癌の診断法
CN101451975B (zh) * 2008-12-29 2012-01-25 浙江大学 一种检测胃癌预后与分期血清蛋白质的方法
EP3157634A4 (fr) * 2014-06-23 2018-02-21 Bionomics, Inc. Anticorps se liant à lgr4

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004004000A1 (fr) * 2002-06-26 2004-01-08 Fujitsu Limited Structure de connexion d'une alimentation a un semi-conducteur
WO2004053066A2 (fr) * 2002-12-06 2004-06-24 Millennium Pharmaceuticals, Inc. Procedes pour identifier, evaluer et traiter des patients suivant une therapie d'inhibition de proteasome

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001053337A1 (fr) * 2000-01-20 2001-07-26 Smithkline Beecham Corporation Recepteur humain axor33 7 fois transmembranaire
EP1290030A2 (fr) * 2000-05-30 2003-03-12 Bayer Aktiengesellschaft Regulation du recepteur couple a la proteine g analogue du lgr-4 humain

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004004000A1 (fr) * 2002-06-26 2004-01-08 Fujitsu Limited Structure de connexion d'une alimentation a un semi-conducteur
WO2004053066A2 (fr) * 2002-12-06 2004-06-24 Millennium Pharmaceuticals, Inc. Procedes pour identifier, evaluer et traiter des patients suivant une therapie d'inhibition de proteasome

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009100737A (ja) * 2007-10-01 2009-05-14 Japan Health Science Foundation α−アクチニン−４遺伝子のコピー数または発現レベルを指標とした癌の診断法
CN101451975B (zh) * 2008-12-29 2012-01-25 浙江大学 一种检测胃癌预后与分期血清蛋白质的方法
EP3157634A4 (fr) * 2014-06-23 2018-02-21 Bionomics, Inc. Anticorps se liant à lgr4

Also Published As

Publication number Publication date
JP4887505B2 (ja) 2012-02-29
JPWO2006112400A1 (ja) 2008-12-11

Similar Documents

Publication Publication Date Title
KR101976219B1 (ko) 유방암의 바이오마커
US20110251097A1 (en) Diagnostic kit of colon cancer using colon cancer related marker and diagnostic method thereof
BRPI0720371B1 (pt) Métodos para diagnóstico ou monitoramento da progressão de câncer de próstata em um indivíduo e uso in vitro de pelo menos um homeodomínio contendo o fator de transcrição como um biomarcador para câncer de próstata
DK2201138T3 (en) GRADING, STAGE DETERMINATION AND FORECAST OF CANCER USING osteopontin-C
EP1493032B1 (fr) Methode de differenciation de metaplasies de lesions neoplasiques ou preneoplasiques
JP5265140B2 (ja) 卵巣癌の検出方法及び検出用キット
WO2012154722A1 (fr) Biomarqueurs prédictifs pour le cancer de la prostate
JP4887505B2 (ja) 癌の転移性及び浸潤能の検査方法
JPWO2019159825A1 (ja) 抗ｐｄ−１抗体／抗ｐｄ−ｌ１抗体療法の効果を予測するバイオマーカー
CN103180455B (zh) 侦测致死系统的方法及其用途
Alba et al. HER2 status determination using RNA-ISH-a rapid and simple technique showing high correlation with FISH and IHC in 141 cases of breast cancer
Chen et al. Fibulin-5 is Down-Regulated in Colorectal Cancer and Correlated with Clinicopathologic Characteristics.
KR102164525B1 (ko) Gdf 15를 포함하는 갑상선 암 진단 또는 갑상선 암 예후 예측용 바이오마커 조성물
CN107022627B (zh) KPNA2基因的应用和抑制KPNA2基因表达的siRNA的应用
JP5574522B2 (ja) 癌マーカー及び癌細胞の検査方法
JP2006520912A (ja) 癌の予後の検知方法
US20100316996A1 (en) Nasopharyngeal cancer malignancy biomarker and method thereof
KR101219979B1 (ko) 갑상선 유두암종의 림프절 전이 진단 방법
KR20120004736A (ko) 갑상선 유두암종의 림프절 전이 진단방법
KR20200025706A (ko) Fgf 21을 포함하는 갑상선 암 진단 또는 갑상선 암 예후 예측용 바이오마커 조성물
US9494594B2 (en) Method of determining administration effect in cancer chemotherapy with S-1
JP2010216826A (ja) 新規腫瘍マーカーを用いた乳癌の検査方法
Zhang et al. Comparison of Her-2/Neu Gene Amplification or Expression between IHC and FISH in BC
JPWO2010117010A1 (ja) 腫瘍マーカーおよびその利用
Monego et al. Borderline HER‐2 breast cancer cases: Histochemical versus real‐time PCR analysis and impact of different cut‐off values

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2007526861

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

NENP Non-entry into the national phase

Ref country code: RU

122 Ep: pct application non-entry in european phase

Ref document number: 06731885

Country of ref document: EP

Kind code of ref document: A1

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载