CN101629958B - Detecting method by TGM2, detecting kit and application thereof - Google Patents
Detecting method by TGM2, detecting kit and application thereof Download PDFInfo
- Publication number
- CN101629958B CN101629958B CN200810132596.5A CN200810132596A CN101629958B CN 101629958 B CN101629958 B CN 101629958B CN 200810132596 A CN200810132596 A CN 200810132596A CN 101629958 B CN101629958 B CN 101629958B
- Authority
- CN
- China
- Prior art keywords
- tgm2
- afp
- serum
- hcc
- biological sample
- 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
- 102100038095 Protein-glutamine gamma-glutamyltransferase 2 Human genes 0.000 title claims abstract description 99
- 101000666171 Homo sapiens Protein-glutamine gamma-glutamyltransferase 2 Proteins 0.000 title claims abstract description 97
- 238000000034 method Methods 0.000 title claims abstract description 34
- 210000002966 serum Anatomy 0.000 claims description 47
- 238000001514 detection method Methods 0.000 claims description 31
- 239000012472 biological sample Substances 0.000 claims description 23
- 239000000523 sample Substances 0.000 claims description 13
- 238000002965 ELISA Methods 0.000 claims description 9
- 230000000052 comparative effect Effects 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims 2
- 208000030062 persistent idiopathic facial pain Diseases 0.000 description 73
- 102100023635 Alpha-fetoprotein Human genes 0.000 description 67
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 55
- 230000014509 gene expression Effects 0.000 description 33
- 206010028980 Neoplasm Diseases 0.000 description 28
- 210000001519 tissue Anatomy 0.000 description 21
- 210000004027 cell Anatomy 0.000 description 18
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 18
- 201000007270 liver cancer Diseases 0.000 description 12
- 208000014018 liver neoplasm Diseases 0.000 description 12
- 108090000623 proteins and genes Proteins 0.000 description 11
- 201000011510 cancer Diseases 0.000 description 10
- 108020004459 Small interfering RNA Proteins 0.000 description 9
- 239000012530 fluid Substances 0.000 description 9
- 102000004169 proteins and genes Human genes 0.000 description 9
- 238000013016 damping Methods 0.000 description 8
- 230000001105 regulatory effect Effects 0.000 description 8
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 7
- 238000012512 characterization method Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 239000012097 Lipofectamine 2000 Substances 0.000 description 5
- 230000006907 apoptotic process Effects 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 238000007654 immersion Methods 0.000 description 5
- 239000013642 negative control Substances 0.000 description 5
- 210000002381 plasma Anatomy 0.000 description 5
- 201000009030 Carcinoma Diseases 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 208000019425 cirrhosis of liver Diseases 0.000 description 4
- 238000003745 diagnosis Methods 0.000 description 4
- 230000036541 health Effects 0.000 description 4
- 238000011532 immunohistochemical staining Methods 0.000 description 4
- 210000004185 liver Anatomy 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- 102000002572 Alpha-Globulins Human genes 0.000 description 3
- 108010068307 Alpha-Globulins Proteins 0.000 description 3
- 102000013529 alpha-Fetoproteins Human genes 0.000 description 3
- 108010026331 alpha-Fetoproteins Proteins 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 230000004069 differentiation Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 208000006454 hepatitis Diseases 0.000 description 3
- 238000003364 immunohistochemistry Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 230000008929 regeneration Effects 0.000 description 3
- 238000011069 regeneration method Methods 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- ZXKXJHAOUFHNAS-FVGYRXGTSA-N (S)-fenfluramine hydrochloride Chemical class [Cl-].CC[NH2+][C@@H](C)CC1=CC=CC(C(F)(F)F)=C1 ZXKXJHAOUFHNAS-FVGYRXGTSA-N 0.000 description 2
- 102000007469 Actins Human genes 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 2
- 208000026310 Breast neoplasm Diseases 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 206010008909 Chronic Hepatitis Diseases 0.000 description 2
- 206010073364 Ductal adenocarcinoma of pancreas Diseases 0.000 description 2
- 206010016654 Fibrosis Diseases 0.000 description 2
- 102000009127 Glutaminase Human genes 0.000 description 2
- 108010073324 Glutaminase Proteins 0.000 description 2
- 108700039882 Protein Glutamine gamma Glutamyltransferase 2 Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000007882 cirrhosis Effects 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 230000009977 dual effect Effects 0.000 description 2
- 230000013020 embryo development Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 210000005229 liver cell Anatomy 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 201000008129 pancreatic ductal adenocarcinoma Diseases 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000007056 transamidation reaction Methods 0.000 description 2
- GHCZTIFQWKKGSB-UHFFFAOYSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;phosphoric acid Chemical compound OP(O)(O)=O.OC(=O)CC(O)(C(O)=O)CC(O)=O GHCZTIFQWKKGSB-UHFFFAOYSA-N 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 102000011727 Caspases Human genes 0.000 description 1
- 108010076667 Caspases Proteins 0.000 description 1
- 102000009058 Death Domain Receptors Human genes 0.000 description 1
- 108010049207 Death Domain Receptors Proteins 0.000 description 1
- 102000004860 Dipeptidases Human genes 0.000 description 1
- 108090001081 Dipeptidases Proteins 0.000 description 1
- 201000009051 Embryonal Carcinoma Diseases 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 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
- 101001044927 Homo sapiens Insulin-like growth factor-binding protein 3 Proteins 0.000 description 1
- 101100152829 Homo sapiens TGM2 gene Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 102100022708 Insulin-like growth factor-binding protein 3 Human genes 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical group OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 125000002842 L-seryl group Chemical group O=C([*])[C@](N([H])[H])([H])C([H])([H])O[H] 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101000666170 Mus musculus Protein-glutamine gamma-glutamyltransferase 2 Proteins 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 102000006010 Protein Disulfide-Isomerase Human genes 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 108010026552 Proteome Proteins 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 241000808793 Strigula Species 0.000 description 1
- 206010043276 Teratoma Diseases 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000005775 apoptotic pathway Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000009087 cell motility Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000013480 data collection Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 231100000234 hepatic damage Toxicity 0.000 description 1
- 231100000753 hepatic injury Toxicity 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000013332 literature search Methods 0.000 description 1
- 230000008818 liver damage Effects 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 201000010893 malignant breast melanoma Diseases 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000003147 molecular marker Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 108020003519 protein disulfide isomerase Proteins 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000003118 sandwich ELISA Methods 0.000 description 1
- -1 simultaneously Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000004960 subcellular localization Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 238000004885 tandem mass spectrometry Methods 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 238000003146 transient transfection Methods 0.000 description 1
- 230000005748 tumor development Effects 0.000 description 1
- 210000001325 yolk sac Anatomy 0.000 description 1
Images
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to a detecting method by using TGM2, a detecting kit and application thereof, in particular to the method for detecting HCC by TGM2 which contains a new mark independently or is combined with AFP, the detecting kit and application thereof.
Description
Technical field
The present invention relates to field of biology, particularly, the present invention relates to use detection method, detection kit and the application thereof of TGM2, in particular to containing the new marker detection HCC of independent use or detecting method, detection kit and the application thereof of the HCC of AFP feminine gender with AFP coupling.
Background technology
Liver cancer (Liver cancer) is a kind of malignant tumour of serious harm human health, and whole world new cases approximately 564,000 people in 2000 ranked fifth position in all malignant tumours.Because its grade malignancy is high, prognosis mala, five year survival rate less than 10%.China is the district occurred frequently of liver cancer, has concentrated the new cases in the whole world approximately 54%, and wherein hepatocellular carcinoma (hepatocellular carcinomas, HCC) accounts for the more than 90% of primary carcinoma of liver
[1,2].According between 1991-2000, the cause of the death sample survey demonstration of China's 169,871 populations, its mortality ratio comes the 2nd of whole malignant tumours, and annual death rate is 54.7/100,000 (man 81.2, female 29.0)
[3].
Alpha-fetoprotein (alpha-fetoprotein, AFP) is the hepatocellular carcinoma mark of approving clinically at present.The 1950's, the people such as Bergstrand have found that this electrophoretic mobility is equivalent to the protein of alpha globulin first in human foetus's serum, its existence do not detected in normal adult serum
[4].Nineteen sixty, Abelev G and colleague in the hepatocellular carcinoma of mouse, found this alpha globulin, it is not present in any tissue of normal mouse, verified it be the principal ingredient in tire mouse serum, when adult rats liver cell regeneration, can reappear
[5].Afterwards, in patients with hepatocellular carcinoma and teratoma patients serum, the specific alpha globulin of embryo was also detected
[6,7].Therefore, people are by this albumen called after alpha-fetoprotein that is mainly present in fetal tissue.
Known at present, AFP is a kind of secreting type glycoprotein, belongs to albumin-like gene family with albumin.It is mainly present in rodent and people's serum embryonic period, embryonic phase, by embryonic development yolk-sac entoderm in period and tire liver, is synthesized and is secreted in blood.In human embryos, AFP maximum concentration can reach 3-4mg/ml, and after birth, in serum, AFP concentration declines rapidly, and neonatal period is about 10-50 μ g/ml, and normal adult is generally below 10ng/ml.In period of gestation maternal serum, the variation of AFP concentration can be pointed out multiple embryonic development defect
[8].And in adult, the serum afp of rising does not exist only in the hepatocellular carcinoma patient of 60%-70%, is found in about 20% chronic hepatitis yet, the hepatitis cirrhosis of 20%-60% and some embryonal carcinoma patient
[9].In having the patients with hepatocellular carcinoma of chronic hepatitis and cirrhosis background, the diagnostic value of AFP is lower
[10,11].
In sum, AFP is for the differentiation sensitivity of patients with hepatocellular carcinoma between 41%-97%, and specificity is at 80%-95%, and positive predictive value is hovered between 9%-58%
[10-12].What is more important, has the patients with hepatocellular carcinoma serum afp of 20%-30% not raise clinically, thereby has greatly limited its using value in hepatocellular carcinoma clinical examination.Therefore, in the urgent need to finding, can combine use with AFP at present, improve the not high hepatocellular carcinoma molecular marker of new mark, especially serum afp of HCC diagnosis accuracy, and the new liver cancer detection method based on described mark and can carry out conveniently detection kit.
Summary of the invention
In order to address the above problem, it is that differential expression protein between HL-7702 is composed that the inventor utilizes the quantitative protein group research strategy of the cold labeling technology combined high precision mass spectrum (nano LTQ-FT MS/MS) of cell in cultivating to analyze two hepatoma cell line HepG2 (AFP is positive) with different AFP expression characterizations and SK-HEP-1 (AFP is negative) and normal liver cell, identify the protein of an obvious high expressed in the SK-HEP-1 of AFP feminine gender hepatoma carcinoma cell, tissue turns glutaminase enzyme 2 (Tissue transglutaminase2, TGM2).This albumen has various isomeric forms, and the expression in SK-HEP-1 is 15 times of HepG2, is 35 times in HL-7702.
TGM2 belongs to and turns glutaminase zymoprotein family, has Ca
2+the transamidation relying on, can be linked to glutaminic acid residue on the lysine epsilon-amino of protein, causes albumen multimerization or protein cross.In addition, TGM2 also has GTP binding ability (sealing its transamidation), and performance G α h protein active, forms the tetramer with G β h and α 1B-adrenocepter, regulates the PLC of receptor activation active.Recently find, TGM2 also has deaminase, dipeptidase, disulfide isomerase activity, TGM2 on human breast cancer cell film also has Ser/Thr protein kinase activity, can be by IGFBP3 (pro-apoptosis bioactivity with the non-dependence of IGF) phosphorylation, thus the Apoptosis signal of induction death receptor intermediary
[13-17].
Therefore, TGM2 is a multifunctional protein molecule, the regulate several biological processes such as the endocytosis of participation Cell Differentiation, apoptosis, cell adhesion, acceptor intermediary and cell movement migration, especially its effect in Apoptosis has dual character, and its concrete effect depends on the apoptosis pathway of special existence in different cell types, the factors such as conversion of the kind of stimulation, Subcellular Localization and the various enzymatic activitys of TGM2
[16,18].Existing document proves, TGM2 is high expressed in breast cancer, melanoma and ductal adenocarcinoma of pancreas, and with chemotherapy resistance and invasion and attack phenotypic correlation
[19-21].In ductal adenocarcinoma of pancreas cell, TGM2 can rely on by AIF, and caspase is non-, and Dependent brings out Apoptosis
[22].Be positioned the TGM2 in tumor stroma, can also suppress growth and metastasis of tumours process
[23,24].Therefore,, in the generation evolution of tumour, TGM2 has various function.For the result of study discovery of liver, TGM2 raises at the early expression of liver fibrosis, but along with course advancement, its expression declines again gradually.In addition,, in liver regeneration, it can also act on α 1B-adrenocepter path
[25-29].
On the basis of above-mentioned discovery, inventor has set up first and this TGM2 molecule has been reduced to method, detection kit and the application thereof of mistaken diagnosis for the auxiliary diagnosis of AFP negative HCC.
Particularly, whether extremely the invention provides the TGM2 level detecting in biological sample to be measured, and then obtain the method as the information of intermediate result, described method comprises step:
1) measure the TGM2 level in biological sample to be checked;
2) the TGM2 level of the TGM2 level in biological sample more to be checked and the contrast of standard biological sample;
3) obtain the information as intermediate result,
4) according to comparative result, determine whether biological sample to be checked is likely HCC sample.
2. 1 method described in, further comprises the joint-detection with AFP.
3. 1 or 2 method, wherein step 1 described in) biological sample be tissue, body fluid or the excreta that has departed from human body or animal body.
4. 1 or 2 method, wherein step 1 described in) biological sample be tissue and serum.
5. 1 or 2 method, wherein step 1 described in) mensuration be the Western marking or immunohistochemistry or ELISA method.
6. 1 method, wherein step 1 described in) mensuration be the mensuration to the downward of TGM2 level.
7. use TGM2 to detect the method for the HCC of AFP feminine gender, described method comprises step:
1) measure the TGM2 level in biological sample to be checked;
2) the TGM2 level of the TGM2 level in biological sample more to be checked and the contrast of standard biological sample;
3) according to comparative result, determine whether biological sample to be checked is HCC sample.
7. 6 method, wherein step 1 described in) biological sample be tissue and serum.
8. 6 method, wherein step 1 described in) mensuration be the Western marking or immunohistochemistry or ELISA method.
9.TGM2 is as the purposes of the mark of HCC.
10. 9 purposes described in, wherein TGM2 and AFP use in conjunction.
The HCC detection kit of the 11. AFP feminine genders for detection of TGM2.
The HCC detection kit purposes of 11 AFP feminine gender described in 12., comprising: as the TGM2 of standard, anti-TGM2 antibody.
The 13. HCC detection kit for joint-detection TGM2 and AFP, comprising: as the TGM2 of standard, anti-TGM2 antibody; AFP, AntiAFP antibody as standard.
The invention provides the method for using TGM2 to detect the negative HCC of AFP, described method comprises step:
1) measure the TGM2 level in biological sample to be checked;
2) the TGM2 level of the TGM2 level in biological sample more to be checked and the contrast of standard biological sample;
3) according to comparative result, determine that biological sample possibility to be checked is HCC sample.
Optionally, the biological sample step 1 of above-mentioned method) is tissue and serum.Optionally, the mensuration step 1 of above-mentioned method) is the Western marking or immunohistochemistry or ELISA method.Optionally, the mensuration of the mensuration step 1 of above-mentioned method) to the downward of TGM2 level.
On the one hand, the invention provides TGM2 as the purposes of the mark of HCC.Optionally, in above-mentioned purposes, TGM2 and AFP use in conjunction.
On the other hand, the invention provides the HCC detection kit for detection of the AFP feminine gender of TGM2, the HCC detection kit for joint-detection TGM2 and AFP is also provided.Described kit, comprising: as the TGM2 of standard, anti-TGM2 antibody; Or wherein also comprise: as the TGM2 of standard, anti-TGM2 antibody; As AFP, the AntiAFP antibody of standard, in described kit, also comprise in addition can for detection of damping fluid well known in the art etc.
Accompanying drawing explanation
The expression of Fig. 1 .TGM2 in hepatocellular carcinoma.Wherein,
(A) the Western marking result of TGM2 in 18 routine liver cancer pairing tissues.In figure, N represents the other normal structure of cancer; C represents tumor tissues.The bar chart on each routine right side represents the relative expression quantity (gray-scale value of TGM2/ β-actin) of TGM2 in the other normal structure of cancer and tumor tissues.(B) the representational immunohistochemical staining figure of TGM2 (200 *).Wherein, 1 and 2 is tumour and the other normal structure of cancer of the negative case of a routine AFP; 3 and 4 is tumour and cancer beside organisms of a routine AFP positive case.
The reticent effect of the specificity of AFP-siRNA in Fig. 2 .HepG2 cell.With 20nM, 50nM, 100nM, 150nM and 200nM AFP-siRNA transfection HepG 2 cell as shown in the figure, harvesting after transfection 72h, through the Western marking, detect the expression of AFP and TGM2.β-actin contrasts as applied sample amount.
The concentration profile of Fig. 3 TGM2 in standard normal healthy controls and HCC patients serum.The longitudinal axis has represented its distribution variation in two groups of crowds, the distribution situation of 50% data in the middle of each box has represented, and the line in the middle of box represents median, upper and lower border represents 75% and 25% percentile.Maximal value and the minimum value of the strigula representative data of box above and below.Round dot represents exceptional value.As can be seen from the figure, HCC patient's serum TG M2 concentration is apparently higher than normal healthy controls.
Embodiment
In order further clearly to set forth the present invention, provide specific embodiments of the invention below, but content of the present invention is not limited to described embodiment, the distortion of equal value of the method for any this law and product and modification etc. all comprise within the scope of the invention.
The Western marking of the relation of embodiment 1:AFP and TGM2 is analyzed
In view of the vital role of TGM2 in tumor development and liver regeneration, liver fibrosis, to it, the expression in liver cancer tissue has carried out the check analysis of large sample amount to inventor.Use Transglutaminase II Ab-1 antibody (U.S. Labvision company, Clone CUB7402, Cat.No.MS-224-P) 18 pairs of paired liver cancer tissues have been done to the analysis of the Western marking, result shows TGM2 up-regulated in the case of 50% (3/6) AFP feminine gender, and 12 routine AFP positive cases all show as down-regulated expression.What is more important, in the negative cases of 6 routine AFP, has 4 examples to show and in tumor tissues, have a plurality of low-molecular-weight isomeric forms, and in 12 routine AFP positive cases, this isomeride exists only in the other normal structures of 9 routine cancers (Figure 1A).Through the accurate probability inspection of Fisher, this phenomenon has significant difference (p=0.0049) between two groups.
The immunohistochemical staining analysis of the relation of embodiment 2:AFP and TGM2
The immunohistochemical staining of 47 routine hepatocellular carcinoma samples (Figure 1B) is found to TGM2 is mainly positioned endochylema and cell membrane.In the case of 29 routine AFP feminine genders, 16 examples show as up-regulated in tumour (55.2%), and in 18 routine AFP positive cases, only have TGM2 up-regulated in tumour of 3 examples.Statistical analysis, the expression of TGM2 is obviously relevant to AFP expression status, its difference in two groups of different AFP expression characterizations has conspicuousness (p=0.0354), still, between the indexs such as its expression and tumor size, differentiation degree, there is no obvious correlativity.
The comprehensive Western marking and immunohistochemical staining result (have 4 routine samples for both share), TGM2 is up-regulated in the negative case of AFP (17/32) of half, and only has 3 routine up-regulateds in 29 routine AFP positive cases.The AFP expression status of this difference and case individuality is relevant (p=0.0034) obviously.And, through Spearman rank correlation, check, in tissue, in TGM2 expression characterization and serum, AFP expression presents a kind of negative correlativing relation (p < 0.0001, related coefficient=0.4693), that is to say that individual Serum AFP expression is lower, in its tissue, more show the trend of TGM2 up-regulated in tumour.In conjunction with existing document, in serum, high-caliber AFP is one of hepatocellular carcinoma independently poor prognosis factor
[30], thinking, TGM2 may bring into play protective effect in hepatic injury, and has certain functional cohesion between itself and AFP.
The checking of the functional dependency between embodiment 3:TGM2 and AFP
For the further functional dependency between proof TGM2 and AFP, inventor's design chemosynthesis for the siRNA of AFP, in the HepG2 of AFP high expressed cell, specific expression of lowering AFP.This siRNA is for the 560-579 position nucleotide of mankind AFP mRNA open reading frame, and its sequence is 5 '-CUU CCU GUA UGC ACC UAC AA-dTdT-3 '.Meanwhile, used the irrelevant random series of a fragment gene group as the negative control of this siRNA, its sequence is 5 '-UUC UCC GAA CGU GUC ACG U-dTdT-3 '..Concrete transfection conditions is as follows:
(1) cell is prepared: the HepG2 cell (hepatoma cell line in the growth period of taking the logarithm, purchased from U.S.'s typical case's culture collection center), trypsinization, density according to 50%～60% is inoculated in 24 orifice plates, every hole adds MEM complete medium containing 10% hyclone, and (MEM fluid nutrient medium originates from U.S. Gibco BRL company, hyclone originates from Austrian PAA company) 500 μ l, mix standby;
(2) transient transfection is prepared: first by above-mentioned siRNA sequence annealing in process (the double-stranded siRNA of 2.5nM adds 125 μ l universal buffering liquid, and 90 ℃ are incubated 2 minutes, naturally cool to after room temperature, and 4 ℃ of placements are spent the night standby).Use LipofectAMINE 2000 (Cat.No.11668-019) transfection reagent of U.S. invitrogen company to carry out the transfection of siRNA, transfection method refers to instructions.During experiment, set up 20nM, 50nM, 100nM, 150nM and 200nM AFP-siRNA group simultaneously, set up three parallel holes, set up negative control simultaneously for every group.In brief, for each hole, the siRNA of different final concentrations is added in 50 μ l 0pti-MEM nutrient culture media, simultaneously, LipofectAMINE 2000 reagent are softly mixed, get 1 μ l LipofectAMINE 2000 reagent and mix at another Guan Zhongyu 50 μ l 0pti-MEM nutrient culture media, incubated at room 5 minutes.The siRNA having diluted is mixed within 30 minutes with the LipofectAMINE 2000 after dilution, put upside down and mix gently, incubated at room 20 minutes.Then the potpourri of the siRNA of variable concentrations and LipofectAMINE 2,000 100 μ l are added in culture hole, the final consumption of LipofectAMINE 2000 in every hole is 0.17%.
(3) cell is at 37 ℃, 5%CO
2cell culture incubator in cultivate 72h, and cell lysis, by Western marking experiment Analysis.
Found that, the AFP-siRNA sequence of design is in the mode of concentration dependent, the expression of effectively lowering AFP in HepG2 cell, and the AFP-siRNA of 200nM can reduce by 50%～60% AFP to express.Meanwhile, find the increase along with AFP-siRNA consumption, the expression of TGM2 increases (Fig. 2) gradually.This result shows, in hepatoma carcinoma cell, AFP may be the negativity regulatory factor of TGM2, and part regulates and controls the expression of TGM2.
The serum ELISA of embodiment 4:TGM2 detects and the clinical detection of the method is applied
Although TGM2 does not have signal peptide and cross-film district, bibliographical information, approximately has the TGM2 of 10-20% to may reside in cell surface and extracellular matrix, therefore, thinks at present, and it can be secreted into extracellular through non-classical secretory pathway
[13].Yet, in the high trust data collection of human normal plasma's albumen producing at human plasma proteomic program (Human Plasma Proteome Project, HPPP) (containing 3020 protein), the existence of this albumen do not detected.Meanwhile, without any TGM2, can appear at the relevant report in normal or various tumor patient blood plasma at present.Expression characterization based on this albumen and the applicant the result in liver cancer tissue, applicant's supposition, TGM2 can be secreted and enter serum/plasma by hepatoma carcinoma cell, and learns as liver cancer early stage blood serum the Specific marker detecting.Therefore, applicant has set up the indirectly two sandwich ELISA detection methods of serum of highly sensitive mankind TGM2 first, and its testing result is evaluated.
The concrete steps of the method are:
1. coated: with 50mM carbonate buffer solution (the 15mM Na of pH9.6
2cO
3; 35mMNaHCO
3) as dilution, by the anti-human TGM2 antibody of goat, (Cat.No.sc-34543) dilution is 2 μ g/ml for TGase2 (N-18), SantaCruz Biotechnology company.In the micropore of 96 hole ELISA Plate of polystyrene, add 50 μ l coating buffers, with preservative film, seal, in 4 ℃ of refrigerators, place spend the night (> 16h).Next day, with PBST damping fluid (137mM NaCl, the 2mM KH of 350 μ l 150mM
2pO
4, 10mM Na
2hPO
4, add 0.05% Tween-20, being adjusted to pH7.4) and immersion type washes plate, 3min * 3 time.
2. sealing: every hole add 300 μ l 2%BSA (Sigma-Aldrich company, Cat.No.A7906), room temperature sealing 4h.With 350 μ l PBST damping fluid immersion types, wash plate, 1min * 3 time.
3. antigen is hatched: in each reacting hole, add the HCC patients serum of 50 μ l, hatch 1.5h for 37 ℃, meanwhile, with lavation buffer solution Surrogate antigen, as negative control, set up 4 negative control holes.Each sample arranges 2 parallel holes.Hatch after end, discard antigen, with 350 μ l PBST damping fluid immersion types, wash plate, 3min * 3 time.
4. detectable antigens reaction: add mouse anti human TGM2 antibody (Lab vision company, Cat.No.MS-224-P) conduct detection antibody, the incubated at room 1h of 0.4 μ g/ml of PBST damping fluid dilution for 50 μ l in each reacting hole.Then discard, with 350 μ l PBST damping fluid immersion types, wash plate, 3min * 3 time.
5. two anti-hatching: in each reacting hole, add the goat anti-mouse IgG of the horseradish peroxidase-labeled of PBST damping fluids dilution in 1: 5000 for 50 μ l (JacksonImmunoReasearch company, Cat.No.115-005-003), incubated at room 1h.Then discard, with 350 μ l PBST damping fluid immersion types, wash plate, 3min * 3 time.
6.TMB (TMB) substrate colour developing: (Sigma-Aldrich company, Cat.No.15053) is dissolved in 5ml absolute ethyl alcohol, is configured to TMB and stores liquid to take 10mg TMB.Then get 0.5ml TMB storage liquid and add 10ml phosphoric acid citric acid substrate buffer solution (51.4mM Na
2hPO
4, 24.3mM citric acid, pH5.0) in, and add the H of 32 μ l0.75%
2o
2mix, be configured to TMB chromophoric solution.In each reacting hole, add 100 μ l TMB chromophoric solutions, mix gently incubated at room 20min.
7. stop: after treating that color is satisfied with and stablizes, every hole adds 100 μ l 2M H
2sO
4solution cessation reaction, and mix gently.
The mensuration of 8.OD value: immediately ELISA Plate is placed in to Model 680 microplate reader (Bio-rad company), OD450/570 dual wavelength reading.
9. the interpretation of result: first determine the reading of 4 negative control holes, average as background reading.For each routine blood serum sample, first calculate variation between the hole of 2 parallel holes mean value of (between hole variation=two hole OD values poor/two hole OD values), for the sample that between those holes, variation value is less than 15%, get the mean value of 2 repetitions as the OD value of this sample; Otherwise, give up this example sample.Then, utilize serum TG M2 concentration difference between the expression normal healthy controls of OD value relative quantification and HCC patient.
Note: above-mentioned experimental technique material therefor, except special indicating, is domestic analytical reagent.
Utilize said method, 31Li health adult (55 years old mean age that applicant matches to age, sex, maximum 68 years old age, minimal ages 36 years old) and 87 routine HCC patient (53 years old mean age, maximum 74 years old age, minimal ages 15 years old) serum TG M2 concentration is measured.Because this systematic comparison that applicant sets up is stable, most case results are all effective.
Experimental result discovery, the concentration median of the serum TG M2 of health adult is 0.0523, is 0～0.1073 between detection zone.And HCC patient's serum TG M2 concentration median is 0.0863, between detection zone, be 0～2.0080.Through Mann-Whitney rank test, both differences have conspicuousness (p=0.0011).Meanwhile, between the serum TG M2 concentration in normal healthy controls and its age, sex, there is no obvious correlativity (p=0.4590, p=0.4287).And in HCC patient, TGM2 serum levels is relevant to histological grade, low differentiation patient's (median is 0.2665) serum-concentration apparently higher than poorly-differentiated cases (median is 0.0618) (p=0.0334).Tumor size is between 3cm to the tumor patient between 5cm (median is 0.1687), and serum TG M2 level apparently higher than small liver cancer patient (median is 0.0685) (p=0.0220).But, the AFP positive and negative HCC patient, serum TG M2 expression does not have notable difference (p=0.8782).The concentration profile of TGM2 in normal healthy controls and HCC patients serum is shown in Fig. 3.
The above results shows, the serum TG M2 ELISA detection method that applicant sets up has reproducible, highly sensitive feature, the serum TG M2 that uses it for 118Li health adult and HCC patient measures and finds, this index has very high detection sensitivity, and in HCC patients serum TGM2 concentration apparently higher than normal healthy controls (with regard to the comparison between median, in tumour, raise at least 1.7 times), show that it may be a very potential serodiagnosis index.And, between the histological grade of its serum expression and tumour and tumor size, there is obvious correlativity.Because, between this index and serum concentration of AFP, there is no correlativity, show that it,, except can combining use with AFP separately for HCC detection, improves diagnosis effect.
The invention provides a kind of liver cancer detection kit, can effectively distinguish normal healthy controls and HCC patient crowd, completed at present clinical testing 118 examples.
In sum, the present invention has studied the expression of TGM2 in Hepatocellular Carcinoma, finds:
(1) TGM2 high expressed in the negative tissue of AFP of half, and in the positive tissue of AFP whole down-regulated expressions almost;
(2) TGM2 has various isomeric forms, and this phenomenon there is no bibliographical information at present.Find, in the tumor tissues of AFP feminine gender, to have more isomeric forms TGM2, and in AFP positive case, these isomeride being mainly arranged in the other normal structure of cancer, there is obvious correlativity in this characteristic and serum afp;
(3) find to have obvious negative correlation between the expression characterization of TGM2 in liver cancer tissue and serum afp, serum afp is lower, and in hepatocellular carcinoma case, the trend of tumor tissues TGM2 high expressed is more obvious;
(4) specificity is lowered after the expression of AFP, and the expression of TGM2 presents increase trend, shows that AFP may be the negative regulate factor of TGM2.
(5) serum ELISA result shows, TGM2 obviously raises in Serum of Cancer Patients, shows that TGM2 may become a kind of new potential blood serum designated object.
In conjunction with the known function of TGM2, think that it brings into play protective effect in course of liver damage, especially at AFP, express in negative hepatocellular carcinoma Carcinogenesis and play a significant role.What is more important, in the hepatocellular carcinoma of different AFP expression status, the expression characterization of TGM2, points out it may develop into the mark of a potential hepatoma carcinoma cell.According to literature search, there is no the report of identical work at present both at home and abroad.
List of references:
1.Parkin DM, Bray F, Ferlay J, Pisani P, global cancer statistics, 2002 (Global cancer statistics, 2002) .CA: clinician's tumour magazine (CA.Cancer J.Clin), 2005; 55 (2): 74-108.
2.Farazi PA, DePinho RA, hepatocellular carcinoma paathogenic factor: from gene to environment (Hepatocellular carcinoma pathogenesis:from genes toenvironment). naturally summarize cancer (Nat Rev Cancer), 2006,6 (9): 674-687.
3.He J, Gu DF, Wu XG, Reynolds K, Duan XF, Yao CH, WangJL, Chen CS, Chen J, Wildman RP, Klag MJ, Whelton PK, Chinese male and women's death Cause Analysis (Major Causes of Death among Men andWomen in China). New England Journal of Medicine (N Engl J Med), 2005; 353:1124-34.
4.Bergstrand CG, Czar B, newfound a kind of protein fragments from human foetus's serum (Demonstration of a new protein fraction in serum fromthe human fetus). the clinical Scandinavia magazine (Scand.J.Clin.Lab.Inves t.) of observing with experiment, 1956; 8:174-179.
5.Abelev GI, Perova S, Khramkova NI, Postnikova ZA, IrlinI, by the embryo source property alpha globulin (Production ofembryonal alpha-globulin by transplantable mouse hepatomas) of transplanting the generation of mouse hepatoma. transplant communique (Transplant.Bull), 1963; 1:74-180.
6.Tatarinov IuS, current known embryo's specific antigen composition in serum human (Current data on embryo-s pecific antigenic components in thehuman blood serum) .Voprosy medit sinsko khimii magazine (Vopr.Med.Khim.), 1964; 10:584-589.
7.Abelev GI, Assecritova IV, KraevSky NA, Perova SD, Perevodchikova NI, the embryo source property serum alpha globulin in cancer patient: diagnostic value.(Embryonal serum alpha-globulin in cancer patients:diagnostic value). international journal of cancer (Int.J.Cancer), 1967; 2:551-558.
8.Mizejewski GJ, the serum alpha Level of Alpha Fetoprotein (Levels of alpha-fetoprotein duringpregnancy and early infancy in normal and disease states) of pregnancy and infantile period under normal and morbid state, gynemetrics's operation magazine (Obstet Gynecol Surv), 2003; 58 (12): 804-826.
9.Fujiyama S, Tanaka M, Maeda S, Ashihara H, Hirata R, Tomita K, the early diagnosis of hepatocellular carcinoma and follow up a case by regular visits to mark and case control (Tumormarkers in early diagnosis, follow-up and management ofpatients with hepatocellular carcinoma). oncology (Oncology), 2002; 62 Suppl 1:57-63.
10.De Masi S, Tosti ME, Mele A, the examination of hepatocellular carcinoma (Screeningfor hepatocellular carcinoma). alimentary canal and liver disease magazine (Dig.LiverDis), 2005; 37 (4): 260-268.
11.Spangenberg HC, Thimme R, Blum HE, the serologic marker thing of hepatocellular carcinoma (Serum markers of hepatocellular carcinoma). liver disease investigation (Semin.Liver Dis), 2006; 26 (4): 385-390.
12.Marrero JA, hepatocellular carcinoma (Hepatocellular carcinoma). the current viewpoint of gastroenterology (Curr.Opin.Gastroenterol), 2003; 19 (3): 243-249.
13.Zemskov EA, Janiak A, Hang J, Waghray A, Belkin AM, the effect (The role oftissue transglutaminase in cell-matrix interactions) of tTG in cell-extracellular matrix interacts. Front Fields of Life Sciences (Front.Biosci.), 2006; 11:1057-1076.
14.Fesus L, Piacentini M, tTG 2: a kind of multifunctional enzyme (Transglutaminase 2:an enigmatic enzyme with diversefunctions) of being confused sample. biological chemistry science trend (Trends Biochem.Sci.), 2002; 27 (10): 534-539.
15.Mangala LS, Mehta K, tTG 2 (Tissue transglutaminase (TG2) in cancer biology) in oncobiology research. experimental tumor progress (Prog.Exp.Tumor Res.), 2005; 38:125-138.
16.Fesus L, Szondy Z, dead transglutaminase 2 (Transglutaminase 2 in the balance of cell death andsurvival) the .FEBS communication (FEBS Lett.) with surviving of statocyte, 2005; 579 (15): 3297-3302.
17.Facchiano F, Facchiano A, Facchiano AM, the effect (The role of transglutaminase-2and its substrates in human diseases) in human diseases of transglutaminase 2 and substrate thereof. bio-science forward position (Front.Biosci.), 2006; 11:1758-1773.
18.Rodolfo C, Mormone E, Matarrese P, Ciccosanti F, FarraceMG, Garofano E, Piredda L, Fimia GM, Malorni W, Piacentini M, tTG 2 is a kind of multi-functional protein (Tissuetransglutaminase is a multifunctional BH3-only protein) that only has BH 3 domains. journal of biological chemistry (J.Biol.Chem.), 2004; 279 (52): 54783-54792.
19.Herman JF, Mangala LS, Mehta K, the meaning (Implications ofincreased tissue transglutaminase (TG2) expression indrug-resistant breast cancer (MCF-7) cells) increasing is expressed in resistance breast cancer cell MCF-7 by tTG 2. oncogene (Oncogene), 2006; 25 (21): 3049-3058.
20.Fok JY, Ekmekcioglu S, Mehta K, the meaning (Implications of tissuetransglutaminase expression in malignant melanoma) that in malignant mela noma, tTG 2 expresses. tumour molecular therapy (Mol Cancer Ther.), 2006; 5 (6): 1493-1503.
21.Verma A, Wang H, Manavathi B, Fok JY, Mann AP, KumarR, Mehta K, tTG expresses in ductal adenocarcinoma of pancreas to be increased and the meaning (Increased expression of tissuetransglutaminase in pancreatic ductaladenocarcinoma and itsimplications in drug resistance and metastasis) in resistance and in shifting. tumor research (Cancer Res.), 2006; 66 (21): 10525-10533.
22.Fok JY, Mehta K, the apoptosis-induced incitant of tTG is expressed and is caused apoptosis of pancreatic cancer cell (Tissue trans glutaminase induces therelease of apoptosis inducing factor and results in apoptoticdeath of pancreatic cancer cells). apoptosis magazine (Apoptosis), 2007; 12 (8): 1455-1463.
23.Xu L, Begum S, Hearn JD, Hynes RO, GPR56, atypical G-G-protein linked receptor conjunctive tissue transglutaminase 2 (TG2) check melanin tumor cell growth and transfer (GPR56, an atypical G protein-coupled receptor, binds tissue transglutaminase, TG2, and inhibits melanoma tumorgrowth and metastasis). institute of NAS periodical (Proc.Natl.Acad.Sci.U.S.A.), 2006; 103 (24): 9023-9028.
24.Mangala LS, Arun B, Sahin AA, Mehta K, the extracellular matrix that tTG brings out changes inhibition tumor invasion (Tissuetransglutaminase-induced alterations in extracellular matrixinhibit tumor invasion). molecular weight tumor (Mol Cancer), 2005; 4:33.
25.Mirza A, Liu SL, Frizell E, Zhu J, Maddukuri S, MartinezJ, Davies P, Schwarting R, Norton P, Zern MA, tTG is regulated by NF-kappaB, and the effect in hepatic injury and liver fibrosis (Arole fortissue transglutaminase in hepatic injury and fibrogenesis, andits regulation by NF-kappaB). U.S.'s physiology magazine (Am.J.Physiol.), 1997; 272 (2Pt1): G281-288.
26.Grenard P, Bresson-Hadni S, El Alaoui S, Chevallier M, Vuitton DA, Ricard-Blum S, extracellular matrix stabilization process in cross-linking reaction participant's liver fibrosis process of transglutaminase intermediary (Transglutaminase-mediatedcross-linking is involved in the stabilization of extracellularmatrix in human liver fibrosis). hepatology magazine (J.Hepatol.), 2001; 35 (3): 367-375.
27.Piacentini M, Farrace MG, Hassan C, Serafini B, AutuoriF, tTG is released into extracellular matrix (' Tissue ' transglutaminase release from apoptotic cells intoextracellular matrix during human liver fibrogenesis) by apoptotic cell in liver fibrosis process. pathology magazine (J.Pathol.), 1999; 189 (1): 92-98.
28.Nardacci R, Lo Iacono O, Ciccosanti F, Falasca L, Addesso M, Amendola A, Antonucci G, Craxi A, Fimia GM, IadevaiaV, Melino G, Ruco L, Tocci G, Ippolito G, Piacentini M, transglutaminase 2 is brought into play protective effect (Transglutaminase type IIplays a protective role in hepatic injury) in course of liver damage. American Journal of Pathology (Am.J.Pathol.), 2003; 162 (4): 1293-1303.
29.Sarang Z, Molnar P, Nemeth T, Gomba S, Kardon T, MelinoG, Cotecchia S, Fesus L, Szondy Z, tTG 2 (TG2) has G protein active, the liver cell of antagonism Fas-intermediary dead (Tissuetransglutaminase (TG2) acting as G protein protects hepatocytesagainst Fas-mediated cell death in mice) in mouse. hepatology (Hepatology), 2005; 42 (3): 578-587.
30.Peng SY, Chen WJ, Lai PL, Jeng YM, Sheu JC, Hsu HC, high-caliber alpha-fetoprotein and clinical stages in hepatocellular carcinoma, early stage recurrence is relevant with poor prognosis: hepatites virus infections, age, meaning (the Highalpha-fetoprotein level correlates with high stage of p53 and beta-catenin sudden change, earlyrecurrence and poor prognosis of hepatocellular carcinoma:significance of hepatitis virus infection, age, p53 andbeta-catenin mutations). international journal of cancer (Int.J.Cancer.), 2004, 112 (1): 44-50.
Claims (6)
- The purposes of 1.TGM2 in the medicine of preparation detection HCC sample, wherein, detects the TGM2 level in biological sample to be measured, and then obtains the method as the information of intermediate result, and described method comprises step:1) measure the TGM2 level in biological sample to be checked;2) the TGM2 level of the TGM2 level in biological sample more to be checked and standard biological sample;3) obtain the information as intermediate result;4) according to comparative result, determine whether biological sample to be checked is likely HCC sample;Wherein, biological sample step 1) is serum.
- 2. the purposes of claim 1, wherein, described method further comprises the use in conjunction with AFP.
- 3. claim 1 or 2 purposes, wherein step 1) mensuration be by the Western marking or ELISA method.
- 4. the purposes of claim 1, wherein step 1) mensuration be TGM2 level and serum afp to be to the mensuration of negative correlativing relation.
- The purposes of 5.TGM2 in the medicine of the blood serum designated object of preparation detection HCC.
- 6. the purposes of claim 5, wherein TGM2 and AFP use in conjunction.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810132596.5A CN101629958B (en) | 2008-07-17 | 2008-07-17 | Detecting method by TGM2, detecting kit and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810132596.5A CN101629958B (en) | 2008-07-17 | 2008-07-17 | Detecting method by TGM2, detecting kit and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101629958A CN101629958A (en) | 2010-01-20 |
| CN101629958B true CN101629958B (en) | 2014-04-09 |
Family
ID=41575137
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200810132596.5A Active CN101629958B (en) | 2008-07-17 | 2008-07-17 | Detecting method by TGM2, detecting kit and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101629958B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140079717A1 (en) * | 2011-02-17 | 2014-03-20 | Cornell University | Compositions and Methods to Prevent Cell Transformation and Cancer Metastasis |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104278013B (en) * | 2013-09-02 | 2017-02-15 | 武汉谐康科技有限公司 | Anti-human tissue transglutaminase 2 monoclonal antibody and application thereof |
| CN110174513A (en) * | 2019-04-15 | 2019-08-27 | 中山大学附属第一医院 | Application of the TGM2 in the biomarker of preparation monitoring Crohn disease disease activity |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1712542A (en) * | 2004-06-25 | 2005-12-28 | 中国科学院上海生命科学研究院 | Screen and use for labelled proto-protein 18 of protein molecule related to hepatocellular carcinoma |
-
2008
- 2008-07-17 CN CN200810132596.5A patent/CN101629958B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1712542A (en) * | 2004-06-25 | 2005-12-28 | 中国科学院上海生命科学研究院 | Screen and use for labelled proto-protein 18 of protein molecule related to hepatocellular carcinoma |
Non-Patent Citations (2)
| Title |
|---|
| Quantitative proteomic signature of liver cancer cells: tissue transglutaminase 2 could be a novel protein candidate of human hepatocellular carcinoma;Sun Yulin等;《Proceedings of the American Association for Cancer Research Annual Meeting》;20080430;第49卷;第934页第2栏#3934 * |
| Sun Yulin等.Quantitative proteomic signature of liver cancer cells: tissue transglutaminase 2 could be a novel protein candidate of human hepatocellular carcinoma.《Proceedings of the American Association for Cancer Research Annual Meeting》.2008,第49卷 |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140079717A1 (en) * | 2011-02-17 | 2014-03-20 | Cornell University | Compositions and Methods to Prevent Cell Transformation and Cancer Metastasis |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101629958A (en) | 2010-01-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Demirag et al. | Expression of Plakophilins (PKP1, PKP2, and PKP3) in breast cancers | |
| US9316654B2 (en) | TAZ/WWTR1 for diagnosis and treatment of cancer | |
| ES2637411T3 (en) | Methods and assays for measuring p95 Y / O p95 in a sample and antibodies specific for p95 | |
| ES2434017T3 (en) | Use of s-ErbB-3 as a cancer marker | |
| Zheng et al. | Endothelial CD146 is required for in vitro tumor-induced angiogenesis: the role of a disulfide bond in signaling and dimerization | |
| Mendelsohn et al. | Snail as a novel marker for regional metastasis in head and neck squamous cell carcinoma | |
| Youns et al. | Serum talin-1 is a potential novel biomarker for diagnosis of hepatocellular carcinoma in Egyptian patients | |
| Demirag et al. | Expression of plakophilins (PKP1, PKP2, and PKP3) in gastric cancers | |
| Kuk et al. | Nidogen-2: a new serum biomarker for ovarian cancer | |
| Pan et al. | ATP synthase ecto-α-subunit: a novel therapeutic target for breast cancer | |
| Guo et al. | High LIFr expression stimulates melanoma cell migration and is associated with unfavorable prognosis in melanoma | |
| Zhuang et al. | Overexpression of Lewis y antigen promotes human epididymis protein 4-mediated invasion and metastasis of ovarian cancer cells | |
| Kim et al. | Elevated expression of thymosin β4, vascular endothelial growth factor (VEGF), and hypoxia inducible factor (HIF)-1α in early-stage cervical cancers | |
| CN102439452A (en) | Methods of determining patient response by measurement of her-2 expression | |
| Tornillo et al. | p130Cas promotes invasiveness of three-dimensional ErbB2-transformed mammary acinar structures by enhanced activation of mTOR/p70S6K and Rac1 | |
| Kolundžić et al. | Galectin-8 is expressed by villous and extravillous trophoblast of the human placenta | |
| Liu et al. | Identification of ANXA1 as a lymphatic metastasis and poor prognostic factor in pancreatic ductal adenocarcinoma | |
| Wu et al. | Fascin1 expression predicts poor prognosis in patients with nasopharyngeal carcinoma and correlates with tumor invasion | |
| Ji et al. | Expression of alpha 1, 6-fucosyltransferase 8 in hepatitis B virus-related hepatocellular carcinoma influences tumour progression | |
| CN101629958B (en) | Detecting method by TGM2, detecting kit and application thereof | |
| Qin et al. | Overexpression of osteopontin promotes cell proliferation and migration in human nasopharyngeal carcinoma and is associated with poor prognosis | |
| Liu et al. | Evaluation of p53 gene expression and prognosis characteristics in uveal melanoma cases | |
| Wan et al. | Six2 is negatively correlated with prognosis and facilitates epithelial-mesenchymal transition via TGF-β/Smad signal pathway in hepatocellular carcinoma | |
| Zheng et al. | FAM98A promotes proliferation of non-small cell lung cancer cells via the P38-ATF2 signaling pathway | |
| US7579158B2 (en) | Cellular retinoic acid binding protein II as a marker for breast cancer |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20151013 Address after: 100021 Beijing, Panjiayuan, South Lane, No. 17, No. Patentee after: Tumor Hospital, Chinese Medical Academy Patentee after: General Hospital of the PLA Navy Address before: 100021 Beijing, Panjiayuan, South Lane, No. 17, No. Patentee before: Tumour Inst., China Medical Science Research Academy Patentee before: General Hospital of the PLA Navy |