CN106932587B - 基于蛋白标志物psg3辅助诊断肝癌或消化道癌症患者的试剂盒 - Google Patents
基于蛋白标志物psg3辅助诊断肝癌或消化道癌症患者的试剂盒 Download PDFInfo
- Publication number
- CN106932587B CN106932587B CN201511020625.5A CN201511020625A CN106932587B CN 106932587 B CN106932587 B CN 106932587B CN 201511020625 A CN201511020625 A CN 201511020625A CN 106932587 B CN106932587 B CN 106932587B
- Authority
- CN
- China
- Prior art keywords
- psg3
- cancer
- detection
- protein
- liver cancer
- Prior art date
- Legal status (The legal status 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 status listed.)
- Active
Links
- 102100022020 Pregnancy-specific beta-1-glycoprotein 3 Human genes 0.000 title claims abstract description 71
- 101000617726 Homo sapiens Pregnancy-specific beta-1-glycoprotein 3 Proteins 0.000 title claims abstract description 70
- 238000003745 diagnosis Methods 0.000 title claims abstract description 21
- 239000012474 protein marker Substances 0.000 title claims abstract description 10
- 206010028980 Neoplasm Diseases 0.000 title abstract description 32
- 201000007270 liver cancer Diseases 0.000 title abstract description 27
- 208000014018 liver neoplasm Diseases 0.000 title abstract description 27
- 201000011510 cancer Diseases 0.000 title description 16
- 206010009944 Colon cancer Diseases 0.000 claims abstract description 26
- 208000001333 Colorectal Neoplasms Diseases 0.000 claims abstract description 19
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 14
- 238000001514 detection method Methods 0.000 claims description 48
- 239000003153 chemical reaction reagent Substances 0.000 claims description 16
- 206010073071 hepatocellular carcinoma Diseases 0.000 claims description 16
- 231100000844 hepatocellular carcinoma Toxicity 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 14
- 230000014509 gene expression Effects 0.000 claims description 9
- 239000003795 chemical substances by application Substances 0.000 claims description 7
- 108091005461 Nucleic proteins Proteins 0.000 claims description 4
- 230000000984 immunochemical effect Effects 0.000 claims description 4
- 102000039446 nucleic acids Human genes 0.000 claims description 4
- 108020004707 nucleic acids Proteins 0.000 claims description 4
- 150000007523 nucleic acids Chemical class 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 3
- 238000011895 specific detection Methods 0.000 claims description 3
- 239000000126 substance Substances 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 abstract description 10
- 208000029742 colonic neoplasm Diseases 0.000 abstract description 8
- 210000005259 peripheral blood Anatomy 0.000 abstract 2
- 239000011886 peripheral blood Substances 0.000 abstract 2
- 108010026331 alpha-Fetoproteins Proteins 0.000 description 15
- 102000013529 alpha-Fetoproteins Human genes 0.000 description 15
- 210000002381 plasma Anatomy 0.000 description 13
- 102000004190 Enzymes Human genes 0.000 description 10
- 108090000790 Enzymes Proteins 0.000 description 10
- 101000617720 Homo sapiens Pregnancy-specific beta-1-glycoprotein 5 Proteins 0.000 description 8
- 239000003550 marker Substances 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 102100022025 Pregnancy-specific beta-1-glycoprotein 5 Human genes 0.000 description 7
- 102000036639 antigens Human genes 0.000 description 7
- 108091007433 antigens Proteins 0.000 description 7
- 101000617725 Homo sapiens Pregnancy-specific beta-1-glycoprotein 2 Proteins 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- 102100022019 Pregnancy-specific beta-1-glycoprotein 2 Human genes 0.000 description 6
- 239000000427 antigen Substances 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 238000002965 ELISA Methods 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 210000004408 hybridoma Anatomy 0.000 description 5
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 4
- XOJVVFBFDXDTEG-UHFFFAOYSA-N pristane Chemical compound CC(C)CCCC(C)CCCC(C)CCCC(C)C XOJVVFBFDXDTEG-UHFFFAOYSA-N 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 208000024891 symptom Diseases 0.000 description 4
- UZOVYGYOLBIAJR-UHFFFAOYSA-N 4-isocyanato-4'-methyldiphenylmethane Chemical compound C1=CC(C)=CC=C1CC1=CC=C(N=C=O)C=C1 UZOVYGYOLBIAJR-UHFFFAOYSA-N 0.000 description 3
- 238000008157 ELISA kit Methods 0.000 description 3
- 101000617728 Homo sapiens Pregnancy-specific beta-1-glycoprotein 9 Proteins 0.000 description 3
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 3
- 102100021983 Pregnancy-specific beta-1-glycoprotein 9 Human genes 0.000 description 3
- 230000002159 abnormal effect Effects 0.000 description 3
- 239000002671 adjuvant Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000001072 colon Anatomy 0.000 description 3
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 201000005202 lung cancer Diseases 0.000 description 3
- 208000020816 lung neoplasm Diseases 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 206010003445 Ascites Diseases 0.000 description 2
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 2
- 101000617708 Homo sapiens Pregnancy-specific beta-1-glycoprotein 1 Proteins 0.000 description 2
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 2
- 206010027476 Metastases Diseases 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 2
- 230000007910 cell fusion Effects 0.000 description 2
- 208000019425 cirrhosis of liver Diseases 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000013399 early diagnosis Methods 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 230000009401 metastasis Effects 0.000 description 2
- 229960000485 methotrexate Drugs 0.000 description 2
- 239000003147 molecular marker Substances 0.000 description 2
- 230000035935 pregnancy Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 2
- 229940104230 thymidine Drugs 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 239000000439 tumor marker Substances 0.000 description 2
- 208000003200 Adenoma Diseases 0.000 description 1
- 206010001233 Adenoma benign Diseases 0.000 description 1
- 238000011725 BALB/c mouse Methods 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 1
- 102100025475 Carcinoembryonic antigen-related cell adhesion molecule 5 Human genes 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 206010008909 Chronic Hepatitis Diseases 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108020004206 Gamma-glutamyltransferase Proteins 0.000 description 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 206010073069 Hepatic cancer Diseases 0.000 description 1
- 101000914324 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 5 Proteins 0.000 description 1
- 101000914321 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 7 Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 108010094028 Prothrombin Proteins 0.000 description 1
- 102100027378 Prothrombin Human genes 0.000 description 1
- MUUGCDGGIVCSDT-UHFFFAOYSA-N [NH4+].[NH4+].[O-]S([O-])(=O)=O.CCCCCCCC(O)=O Chemical compound [NH4+].[NH4+].[O-]S([O-])(=O)=O.CCCCCCCC(O)=O MUUGCDGGIVCSDT-UHFFFAOYSA-N 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000003453 ammonium sulfate precipitation method Methods 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000002591 computed tomography Methods 0.000 description 1
- 230000009274 differential gene expression Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 102000006640 gamma-Glutamyltransferase Human genes 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 239000000155 melt Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000000505 pernicious effect Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 208000014081 polyp of colon Diseases 0.000 description 1
- 230000002980 postoperative effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 235000019624 protein content Nutrition 0.000 description 1
- 229940039716 prothrombin Drugs 0.000 description 1
- 210000000664 rectum Anatomy 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000010183 spectrum analysis Methods 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 208000024719 uterine cervix neoplasm Diseases 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Pathology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Hospice & Palliative Care (AREA)
- Oncology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一种基于蛋白质标记物PSG3辅助诊断癌症,尤其是肝癌与结肠癌的试剂盒。本发明要求保护用于检测蛋白质PSG3的产品在制备用于诊断肝癌与结肠癌的试剂盒中的应用。在本发明中,发明人首次在肝癌和结肠癌患者外周血中发现了蛋白标记物PSG3的存在,并发现肝癌和结肠癌患者外周血中蛋白标记物PSG3的浓度高于健康者,即蛋白标记物PSG3具有辅助诊断肝癌和结肠癌患者的价值。本发明对肝癌和结肠癌的辅助诊断具有重要意义。
Description
技术领域
本发明涉及一种蛋白标志物PSG3在辅助诊断肝癌或消化道患者中的应用,以及制备相应的检测试剂盒中的应用。
背景技术
原发性肝癌是全球第6位、中国第3位最常见的癌症。据国际癌症研究中心(IARC)估计,2000年全球肝癌发病数为56.4万人,其中约55%发生在中国,即中国肝癌发病30.6万人,肝癌死亡30.0万,占我国居民肿瘤死亡的第二位。肝癌出现症状时多属中晚期,切除后复发、转移率高。因此,肝癌的早期诊断对延长患者的生存时间和降低肝癌死亡率具有重要意义。
目前,影像学诊断、细胞与组织学诊断及化学诊断是肿瘤诊断的三大主要方法。影像学诊断在肝癌诊断中起重要的作用,但是在诊断小肝癌及区分良恶性结节中均具有一定的局限。超声检查或CT阳性结果,结合血清甲胎蛋白(α-fetoprotein,AFP)水平高于400ng/ml的检测结果,可对肝癌做出确诊。但是,通常当这些条件都符合时,已经错过了治疗的最佳时机。无论是超声波检查、CT扫描还是核磁共振,对小病灶的鉴定都有一定的局限性,尤其是肝硬化结节在影象学上与小肝癌结节有许多的相似之处。因此,虽然影像学技术取得了巨大的进步,但临床上仍需结合肝癌的分子标志物,在较为复杂的病例中将良恶性肝病进行区分。此外,肿瘤的临床分期及肿瘤大小是肝癌治疗效果的决定因素,因此,肿瘤标志物还可用于肝癌危险人群(如HBV慢性携带者和肝硬化患者)的普查。
目前肝癌诊断最常用的标志物是甲胎蛋白(AFP),在症状和影像学改变发生前数月即可出现异常。正常人血清中AFP含量一般小于10ng/ml。当AFP的诊断值定为20ng/ml时,其敏感性为50~60%,但在肿瘤较小的病例中,敏感性显著降低,有报道仅为40%。单独使用AFP作为肝癌诊断标志物的另一个问题是缺乏特异性,在相当多的慢性肝炎患者尤其是肝硬化患者中,AFP含量也达20-200ng/ml。目前认为可与AFP互补诊断的标志物有AFP异质体、γ-谷氨酰转移酶同工酶II和异常凝血酶原等,但由于它们在敏感性和特 异性上的不足,以及检测方法的繁琐复杂,使其始终停留在实验室研究水平,未能转化成临床常规检查项目。因此,探索新的肿瘤分子标志物,以及建立相应的易于推广的检测方法,仍是当今肝癌研究领域的重要课题之一。
同样,消化道肿瘤在我国也是导致死亡的主要病因之一。其中的结直肠癌起病隐匿,早期常无明显的临床症状,而且病情发展较慢,病人出现明显症状时大多已经到了中晚期。目前,结肠的治疗主要是以手术为主,但结肠癌转移后的化疗效果差,术后约有40%的患者出现复发,且晚期患者术后生存率较低。尽管有许多学者已经做了大量的关于结肠癌分子水平的研究,但是目前仍然缺乏有效地早期诊断结肠癌的分子标志物。肿瘤标志物可以有效地作为临床诊断依据,可以监测高危人群、早期诊断、指导治疗、判断治疗疗效、检测复发及转移,是肿瘤患者的重要检查指标、具有重要的临床意义。结肠癌的发生前期会有一段时间的癌前病变,如结肠息肉,因此在早期发现并阻断或延迟癌症的发展,有极其重要的意义。因此,寻找发现有价值的肿瘤标志物对于提高消化道肿瘤、尤其是结直肠癌的诊断和预测发生、发展及改善预后具有重要的临床指导意义。
人类血液中包含丰富的各种细胞成分和分子物质,能很好地反映机体不同组织和器官的生理、病理状态,且其标本容易获得,因此,是一种理想的肿瘤无创诊断手段。因此,仍有待开发敏感性和特异性良好的标志物。
妊娠特异蛋白3(Human pregnancy specific beta-1-glycoprotein 3,PSG3),是一种分泌到细胞外的糖蛋白。PSG家族中共包含11个蛋白,包括PSG1、PSG2、PSG3等。PSG蛋白一般都包括N端同免疫球蛋白可变区类似的结构,随后是2-3个免疫球蛋白恒定区C2样结构域,以及一个短的疏水尾,位于C端。PSG根据其结构组成可分为4类,I型(N-A1-A2-B2-C)、IIa型(N-A1-B2-C)和IIb型(N-A2-B2-C)、III型(N-B2-C)、IV型(A1-B2-C)。目前PSG家族的作用机制尚不清楚,在正常情况下,胎盘的合胞体滋养层细胞中大量表达,在妊娠期女性外周血中PSG蛋白含量升高,可达200-400μg/ml。关于PSG家族在癌症中的研究鲜有报道。
PSG3蛋白长度428个氨基酸,分子量47,945Da,蛋白的氨基酸序列如下(SEQ IDNO:1):
MGPLSAPPCT QRITWKGLLL TALLLNFWNL PTTAQVTIEA
EPTKVSKGKD VLLLVHNLPQ NLAGYIWYKG QMKDLYHYIT
SYVVDGQIII YGPAYSGRET VYSNASLLIQ NVTREDAGSY
TLHIVKRGDG TRGETGHFTF TLYLETPKPS ISSSNLYPRE
DMEAVSLTCD PETPDASYLW WMNGQSLPMT HSLQLSKNKR
TLFLFGVTKY TAGPYECEIR NPVSASRSDP VTLNLLPKLP
KPYITINNLN PRENKDVLAF TCEPKSENYT YIWWLNGQSL
PVSPRVKRPI ENRILILPSV TRNETGPYQC EIQDRYGGIR
SYPVTLNVLY GPDLPRIYPS FTYYHSGENL YLSCFADSNP
PAEYSWTING KFQLSGQKLF IPQITTKHSG LYACSVRNSA
TGMESSKSMT VKVSAPSGTG HLPGLNPL
有关PSGs在肿瘤中的研究报道较少。Salahshor等应用差异基因表达谱分析发现结直肠癌、肝转移癌、子宫肿瘤组织中均可检测到异常升高的PSG9转录本,与正常结直肠粘膜相比,PSG9在腺瘤中上调2倍,且结直肠癌PSG9表达异常上调呈APC突变依赖性(Salahshor et al,2005)。Kamarli ZP等的研究表明,PSG1在骨恶性肿瘤患者血清中蛋白浓度升高(Kamarli et al,2004)。而关于PSG3在肿瘤中的研究还未见报道。
发明内容
本发明第一方面涉及检测PSG3蛋白的分子在制备用于辅助诊断癌症,尤其是肝癌或消化道癌症患者的试剂盒或检测试剂中的用途。
根据本发明第一方面任一项的用途,其中所述检测PSG3蛋白的分子是指能够特异性检测PSG3蛋白表达的分子,例如可以为核酸、蛋白质或化合物。
根据本发明第一方面任一项的用途,其中所述蛋白质为抗体,其能够与PSG3蛋白特异性结合,优选为单克隆抗体。
在本发明的实施方案中,所述检测PSG3蛋白的分子为单克隆抗体,其能够与PSG3蛋白特异性结合。
在本发明的具体实施方案中,采用免疫化学的方法检测PSG3蛋白。
根据本发明第一方面任一项的用途,其中所述的肝癌为肝细胞肝癌。
根据本发明第一方面任一项的用途,其中所述的消化道癌症为结直肠癌。
根据本发明第一方面任一项的用途,所述试剂盒或检测试剂用于血浆样 品的检测。
根据本发明第一方面任一项的用途,其中所述辅助诊断的判断方法为,血浆中PSG3的含量高于551.65μg/ml,则待检者为候选的肝细胞肝癌或结直肠癌患者。
根据本发明第一方面任一项的用途,其中所述检测PSG3蛋白的分子还带有可检测的标记。
根据本发明第一方面任一项的用途,其中所述试剂盒或检测试剂中还含有缓冲液及显色剂。
根据本发明第二方面涉及一种用于辅助诊断癌症,尤其是肝癌或消化道癌症的试剂盒或检测试剂,其含有检测标记物PSG3蛋白的产品。
根据本发明第二方面涉及一种用于辅助诊断癌症,尤其是肝癌或消化道癌症的试剂盒或检测试剂,所述检测标记物PSG3蛋白的产品是指能够特异性检测PSG3蛋白表达的分子,例如可以为核酸、蛋白质或化合物。
根据本发明第二方面任一项的试剂盒或检测试剂,其中所述蛋白质为抗体,其能够与PSG3蛋白特异性结合,优选为单克隆抗体。
在本发明的实施方案中,所述检测PSG3蛋白的分子为单克隆抗体,其能够与PSG3蛋白特异性结合。
在本发明的具体实施方案中,采用免疫化学的方法检测PSG3蛋白。
根据本发明第二方面任一项的试剂盒或检测试剂,其中所述的肝癌为肝细胞癌,所述的消化道癌症为结直肠癌。
根据本发明第二方面任一项的试剂盒或检测试剂,所述试剂盒或检测试剂用于血浆样品的检测,优选地,所述检测方法为免疫化学方法。
根据本发明第二方面任一项的试剂盒或检测试剂,其中所述辅助诊断的判断方法为,血浆PSG3蛋白表达高于551.65μg/ml,则待检者为候选的肝细胞肝癌或结直肠癌患者。
根据本发明第二方面任一项的试剂盒或检测试剂,其中所述检测PSG3蛋白的分子还带有可检测的标记。
根据本发明第二方面任一项的试剂盒或检测试剂,其中所述试剂盒或检测试剂中还含有缓冲液及显色剂。
在本发明中,其中所述检测PSG3蛋白的分子是指能够检测PSG3蛋白是否过表达的分子,例如可以为核酸、蛋白质或化合物。
在本发明中,可以采用本领域公知的方法制备抗体。
可以使用免疫测定的方法定量或定性检测蛋白标志物的存在。所述免疫测定通常包括将生物样品与抗体一起孵育,并通过多种熟知技术检测结合抗体。
在本发明中,所述化合物具有本领域公知的含义,其可以和PSG3蛋白特异性结合,进而用于检测PSG3蛋白的表达。
在本发明中,所述检测PSG3蛋白的分子上可以连接有可检测的标记分子。
在本发明中,所述试剂盒或检测试剂中还可以包含能够与检测PSG3蛋白的分子结合的分子,例如抗体,例如第二抗体,该分子也可以连接有可检测的标记分子。
在本发明中,所述可检测的标记分子例如可以为酶、荧光素、金属离子或同位素等。
附图说明
图1A为PSG3区分正常人和肝细胞肝癌患者(HCC)的ROC曲线。
图1B为PSG3区分正常人和结直肠癌患者(CC)的ROC曲线。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1制备PSG3检测的ELISA试剂盒
采用大肠杆菌原核表达PSG3蛋白,表达载体为pET30a。取5-6周龄BALB/c小鼠,利用PSG3全长蛋白作为抗原添加弗氏佐剂免疫制备PSG3抗体。每只小鼠免疫4次,每次间隔2周,每次利用15-30μg抗原蛋白,第一次免疫注射时利用完全佐剂,后三次利用不完全佐剂。细胞融合前三天,从三只脾细胞供体小鼠挑选一只进行静脉注射PSG3抗原做进一步免疫。细胞融合、杂交瘤筛选均依据J.M.Davis的方法(Davis et al 1982)。简略过程如下: 加强免疫后的小鼠脾细胞和SP2/0骨髓瘤细胞混合,慢慢加入促融合剂50%聚乙二醇0.7ml。37℃培养1min,10ml IMDM培养基稀释,低速离心。然后用40ml含1.25%甲基纤维素、25%胎牛血清、2%HAT(含次黄嘌呤、甲氨蝶呤和胸腺嘧啶核苷)的IMDM培养基重悬。上下颠倒混合,转移到35mm平板中,每板约2ml,于37℃、5%CO2环境中培养。7天后克隆转移入96孔板,加入含15%胎牛血清和2%HT(含次黄嘌呤和胸腺嘧啶核苷)的IMDM培养基再次培养。当细胞融合度达到50%~70%,收集培养基上清,利用ELISA和Western blot检测是否存在抗体。
建立检测PSG3抗原的酶标双抗体夹心法。检测PSG3抗原的酶标双抗体夹心法的建立:首先利用小鼠腹腔生产单克隆抗体,将得到的阳性杂交瘤细胞分别注入小鼠腹腔,使其产生腹水,注射降植烷,每只小鼠0.5ml;注射降植烷1周后,注射杂交瘤细胞,每只小鼠注射106个细胞。7-10天后分别收集腹水,用正辛酸-硫酸铵沉淀法分别提纯抗体,再用过碘酸钠法将抗体分别进行辣根过氧化物酶标记(酶标抗体)。用纯化的单克隆抗体分别包被酶标板,并分别与酶标抗体配对检测PSG3抗原,获得最佳配对的包被抗体P201以及酶标捕获抗体P209。其中包被抗体P201和酶标的捕获抗体P209分别由杂交瘤细胞株P201和P209制备产生。杂交瘤细胞系P201和P209已于2015年11月27日在中国微生物菌种保藏管理委员会普通微生物中心保藏,保藏号分别为CGMCC NO.11597和CGMCCNO.11598,保藏地址为北京市朝阳区北辰西路一号院3号。
ELISA步骤如下:将PSG3包被抗体P201包被96孔酶标板,100μl/孔,4℃过夜;甩去包被抗体,0.5%PBST洗板3次,加入2%BSA封闭液, 室温4小时;甩去封闭液,0.5%PBST洗板3次,酶标板中每孔分别加入PSG3标准蛋白和待测样本,100μl/孔;再加入PSG3酶标抗体P209,100μl/孔,混匀后4℃过夜;甩去反应液体,0.5%PBST洗板5次, 加入TMB显色液,200μl/孔,避光反应15分钟;加入2M硫酸终止反应,用酶标仪OD450nm处检测吸光度;利用标准蛋白吸光度的值绘制标准曲线,并计算待测样本中PSG3的蛋白浓度。
实施例2在大规模的肿瘤患者和正常人群血浆样本中检测PSG3的蛋 白浓度
利用制备的ELISA试剂盒在怀孕妇女血浆中检测PSG3蛋白的浓度。检测的样本包括:11例怀孕早期妇女(11-12周),17例怀孕晚期妇女(28-38周)。ELISA的结果如表1显示,PSG3在怀孕早期妇女血浆中的蛋白浓度显著高于怀孕晚期(P<0.001)。
表1.PSG3在怀孕妇女血浆中的蛋白浓度(μg/ml)
利用制备的ELISA试剂盒在大规模的肿瘤患者和正常人群血浆样本中检测PSG3的蛋白浓度。检测的样本包括:178例肝细胞肝癌(Hepatocarcinoma,HCC),121例肺癌(Lungcancer,LC),159例卵巢癌(Ovarian Cancer,OC),88例结直肠癌(Colorectal Cancer,CC),以及115例年龄与性别比例匹配的健康正常人(Health Control)。ELISA的结果显示,PSG3在肝细胞肝癌、结直肠癌患者和肺癌患者的血浆蛋白水平显著高于正常对照组(P<0.001,表2)。
表2.PSG3在各组肿瘤患者和正常人群血浆中的蛋白浓度(μg/ml)
实施例3 PSG3血浆蛋白浓度在辅助诊断肝细胞癌或消化道癌症中的作用
当PSG3血浆蛋白浓度值为551.65μg/ml时,检测肝细胞肝癌的敏感性为64.2%,特异性为74.8%,ROC曲线下面积为0.774,95%CI=0.719-0.828,P<0.001(图1A)。同一组肝癌病人血浆中也检测了肝细胞肝癌标志物甲胎蛋白(alpha-fetal protein,AFP)的水平。当使用临床常规检测限值为25ng/ml 时,肝细胞肝癌AFP阴性的病人为107例,占标本总数的60.1%(107/178)。而在此107例AFP阴性的肝癌病人中,PSG3阳性(血浆中PSG3蛋白浓度>551.65μg/ml)的病例数为66例,提高了肝癌病人的检出率,临床上可以辅助肝细胞肝癌病人的诊断率。
当PSG3血浆蛋白浓度值为551.65μg/ml时,检测结直肠癌(n=88)的敏感性为92.0%,特异性为74.8%,ROC曲线下面积为0.873,95%CI=0.819-0.828,P<0.001(图1B)。这88例病人血浆也检测了结直肠癌标志物癌胚抗原(carcinoembryonic antigen,CEA)的蛋白水平。当使用临床常规检测限值为5ng/ml时,结直肠癌患者中CEA阴性的病人为67例,占标本总数的76.1%(67/88)。而在此67例CEA阴性的结直肠癌病人中,PSG3阳性(血浆中PSG3蛋白浓度>551.65μg/ml)的病例数为60例,显著的提高了结直肠癌病人的检出率,临床上有较好的应用前景。
参考文献
1.Bebo,B.F.,Jr.and G.S.Dveksler(2005)."Evidence that pregnancyspecific glycoproteins regulate T-Cell function and inflammatory autoimmunedisease during pregnancy."Curr Drug Targets Inflamm Allergy 4(2):231-237.
2.Blois,S.M.,et al.(2013)."Pregnancy-specific glycoprotein 1(PSG1)activates TGF-[beta]and prevents dextran sodium sulfate(DSS)-induced colitisin mice."Mucosal Immunol.
3.Camolotto,S.,et al.(2010)."Expression and transcriptionalregulation of individual pregnancy-specific glycoprotein genes indifferentiating trophoblast cells."Placenta 31(4):312-319.
4.Fialova,L.,et al.(1991)."[Serum levels of trophoblast-specificbeta-1-globulin(SP1)and alpha-1-fetoprotein(AFP)in pregnant women withrheumatoid arthritis]."Cesk Gynekol 56(3):166-170.
5.Ha,C.T.,et al.(2005)."Binding of pregnancy-specific glycoprotein17to CD9on macrophages induces secretion of IL-10,IL-6,PGE2,and TGF-β1."Journal of Leukocyte Biology 77(6):948-957.
6.Ha,C.T.,et al.(2008)."N-glycosylation is required for binding ofmurine pregnancy-specific glycoproteins 17and 19to the receptor CD9."Am JReprod Immunol 59(3):251-258.
7.Kamarli Z.P.,et al.(2004)."Use of immunoglobulin E and pregnancy-specific beta-1-glycoprotein in differential diagnosis of bone malignancies".Vopr Onkol.50(3):316-319.
8.Lisboa,F.A.,et al.(2011)."Pregnancy-specific Glycoprotein 1 InducesEndothelial Tubulogenesis through Interaction with Cell SurfaceProteoglycans."Journal of Biological Chemistry 286(9):7577-7586.
9.Martinez,F.F.,et al.(2012)."Pregnancy-specific glycoprotein 1aactivates dendritic cells to provide signals for Th17-,Th2-,and Treg-cellpolarization."Eur J Immunol 42(6):1573-1584.
10.McLellan,A.S.,et al.(2005)."Structure and evolution of the mousepregnancy-specific glycoprotein(Psg)gene locus."Bmc Genomics 6:4.
11.Motrán,C.C.,et al.(2003)."In vivo expression of recombinantpregnancy-specific glycoprotein□1a induces alternative activation ofmonocytes and enhances Th2-type immune response."Eur J Immunol 33(11):3007-3016.
12.Pihl,K.,et al.(2009)."First trimester maternal serum pregnancy-specific beta-1-glycoprotein(SP1)as a marker of adverse pregnancy outcome."Prenat Diagn 29(13):1256-1261.
13.Rutherfurd,K.J.,et al.(1995)."A motif in PSG11s mediates bindingto a receptor on the surface of the promonocyte cell line THP-1."MolEndocrinol9(10):1297-1305.
14.Snyder,S.K.,et al.(2001)."Pregnancy-specific glycoproteinsfunction as immunomodulators by inducing secretion of IL-10,IL-6 and TGF-beta1 by human monocytes."Am J Reprod Immunol 45(4):205-216.
15.Salahshor,S.,et al.(2005)."Differential gene expression profilereveals deregulation of pregnancy specific beta1 glycoprotein 9 early duringcolorectal carcinogenesis."Bmc Cancer 5:66.
16.Thompson,J.A.,et al.(1991)."Carcinoembryonic antigen gene family:molecular biology and clinical perspectives."J Clin Lab Anal 5(5):344-366。
Claims (8)
1.用于检测血浆样本中蛋白标志物PSG3浓度的产品在制备用于辅助诊断肝细胞肝癌和结直肠癌患者的试剂盒中的应用。
2.权利要求1所述的应用,其中所述检测使用免疫化学方法进行。
3.权利要求1所述的应用,其中检测蛋白标志物PSG3浓度的产品是指能够特异性检测PSG3蛋白表达的分子。
4.权利要求3所述的应用,其中检测蛋白标志物PSG3浓度的产品为核酸、蛋白质或化合物。
5.权利要求3所述的应用,其中所述特异性检测PSG3蛋白表达的分子为抗体。
6.权利要求5所述的应用,其中所述抗体为单克隆抗体。
7.权利要求3或5所述的应用,其中所述检测PSG3蛋白表达的分子还带有可检测的标记。
8.权利要求7所述的应用,其中所述试剂盒或检测试剂中还含有缓冲液及显色剂。
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201511020625.5A CN106932587B (zh) | 2015-12-29 | 2015-12-29 | 基于蛋白标志物psg3辅助诊断肝癌或消化道癌症患者的试剂盒 |
PCT/CN2016/081858 WO2017113565A1 (zh) | 2015-12-29 | 2016-05-12 | 基于蛋白标志物psg3辅助诊断肝癌或消化道癌症患者的试剂盒 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201511020625.5A CN106932587B (zh) | 2015-12-29 | 2015-12-29 | 基于蛋白标志物psg3辅助诊断肝癌或消化道癌症患者的试剂盒 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN106932587A CN106932587A (zh) | 2017-07-07 |
CN106932587B true CN106932587B (zh) | 2018-07-27 |
Family
ID=59442314
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201511020625.5A Active CN106932587B (zh) | 2015-12-29 | 2015-12-29 | 基于蛋白标志物psg3辅助诊断肝癌或消化道癌症患者的试剂盒 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN106932587B (zh) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110763845B (zh) * | 2018-07-27 | 2022-10-28 | 成都市第三人民医院 | 一种检测蛋白标志物的配体在制备用于诊断结肠癌的产品中的用途和试剂盒 |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE10225677A1 (de) * | 2001-06-11 | 2003-01-16 | Honda Motor Co Ltd | Insassen-Rückhaltesystem |
US20070160601A1 (en) * | 2005-12-09 | 2007-07-12 | Angela Widom | Neutralizing antibodies against primate psgl-1 and uses therefor |
CA2639070A1 (en) * | 2006-04-13 | 2007-11-01 | Oncomethylime Sciences S.A. | Novel tumour suppressor |
CN104151407B (zh) * | 2014-08-01 | 2016-11-23 | 首都医科大学附属北京朝阳医院 | 一种制备人妊娠特异性糖蛋白9的方法 |
-
2015
- 2015-12-29 CN CN201511020625.5A patent/CN106932587B/zh active Active
Also Published As
Publication number | Publication date |
---|---|
CN106932587A (zh) | 2017-07-07 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
BRAUNSTEIN et al. | Ectopic production of human chorionic gonadotrophin by neoplasms | |
JP5711196B2 (ja) | 卵巣癌を判定するためのhe4及び他の生化学マーカーの使用 | |
Liu et al. | Diagnostic value of serum glypican-3 alone and in combination with AFP as an aid in the diagnosis of liver cancer | |
CN101449163B (zh) | S100a12蛋白作为结肠直肠癌标记的用途 | |
Tang et al. | The sensitivity and specificity of serum glycan-based biomarkers for cancer detection | |
CN102504027A (zh) | 一种多表位tk1抗体的制备及其在人群体检筛查中早期肿瘤检测和风险预警中的应用 | |
Wang et al. | Expression of oncofetal antigen glypican-3 associates significantly with poor prognosis in HBV-related hepatocellular carcinoma | |
Bertok et al. | Identification of whole-serum glycobiomarkers for colorectal carcinoma using reverse-phase lectin microarray | |
Wang et al. | Joint effect of THBS2 and VCAN accelerating the poor prognosis of gastric cancer | |
CN106932587B (zh) | 基于蛋白标志物psg3辅助诊断肝癌或消化道癌症患者的试剂盒 | |
CN103954761B (zh) | 用于卵巢癌早中期快速诊断试剂盒及其检测方法 | |
CA2682132C (en) | Use of he4 for assessment of breast cancers | |
CN106928352A (zh) | 一种抗psg3蛋白的单克隆抗体及其杂交瘤细胞株与应用 | |
WO2013063876A1 (zh) | 一种用于检测非小细胞肺癌的双抗体夹心elisa试剂盒及其制备方法 | |
EP3193173A1 (en) | Serological autoantibodies as biomarker for colorectal cancer | |
Huang et al. | Investigation of circulating antibodies to ANXA1 in breast cancer | |
CN103487580A (zh) | Dkk1作为诊断标志物的用途 | |
ES2285469T3 (es) | Utilizacion de la proteina celular transportadora de acido retinoico de tipo ii (crabp ii) como marcador del cancer de mama. | |
CN105037534B (zh) | 一种检测肺癌标志物myc抗原表位氨基酸序列及应用 | |
CN108732354A (zh) | Rcan1.4作为诊断肝细胞癌的诊断标志物的应用 | |
CN105111297B (zh) | 一种检测肝癌标志物imp1抗原表位氨基酸序列及应用 | |
CN105017405B (zh) | 一种检测肝癌标志物bmi1抗原表位氨基酸序列及应用 | |
WO2017113565A1 (zh) | 基于蛋白标志物psg3辅助诊断肝癌或消化道癌症患者的试剂盒 | |
CN110456072A (zh) | 胰岛再生原蛋白1α的应用及其检测方法 | |
El Hagary | Study of the Serum Level of Annexin A5 as a Marker for Hepatocellular Carcinoma in Hepatitis C Virus Cirrhosis Patients |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |