| Primary liver cancer (PLC) is one of the most common malignancy tumor in the world. Early detection with early treatment is very important for prolonging life. alpha-fetoprotein (AFP) is the most popular biomarker that has been widely use for PLC currently. Although AFP can not correctly detect all disorders[1-5], no other biomarkers can take the place of it. The new tumor markers is the goal that people are searching for. In 1996, Pilia et al[6] reported that glypican-3 (GPC3) , which encodes one member of the glypican family, is mutated in patients with Simpson-Golabi-Behmel syndrome. The lately reports indicated that GPC3 mRNA is highly expressed in PLC, especially in small PLC and AFP-negative PLC patients. It suggested that GPC3 mRNA may prove to be an potential novel tumor marker for PLC[7-9]. Our work is to study the possibility for GPC3 protein to be the immunohistochemical and serum marker for PLC and to value its clinical application.The first part of our study is preparation, purification and identification of anti-GPC3 polyclonal antibody and its initial application. Based on our previous study, we induced the GPC3 protein containing 523 amino acid polypeptide by E.coli expressing system. We got ideal antigen by simple method and then prepared rabbit anti-human GPC3 polyclonal antibody. This polyclonal antibody has high titer identified by indirect enzyme-linked immunosorbent assay (ELISA). We purchased protein G latexes to purify this polyclonal antibody and electrophoresed on SDS-PAGE gel stained by Coomassie Brilliant Blue to identify its effect. We then identified the purified antibody by Western Blot analysis. Using pre-immuned antibody and post- immuned antibody, we hybridized hepatocellular cacinoma (HCC) cancerous liver tissues and its corresponding non-cancerous liver tissues. Remarkable difference was found in these two different antibodys. Comparing with pre-immuned antibody, the post-immuned antibody hybridized a main protein band molecular weight of 70kDa which corresponding to GPC3 core protein.The sameband observed in HCC cell lines HepG2, HuH-7> Hep3B but not the non-HCC cell lines Hela. Also by Western Blot analysis, we studied some normal liver tissues around haemangioma, HCC ^ intrahepatic cholangiocellular carcinoma (ICC) cancerous liver tissues and its corresponding non-cancerous liver tissues. It was found that normal liver tissues do not express GPC3 protein. In 26 HCC patients, the percentage of GPC3 protein expressing in cancerous liver tissues and non-cancerous liver tissues is 84.6% (22/26) and 30.8% (8/26), respectively. Among 7 ICC patients, only 2 patients could be observed GPC3 protein expression.The second part of our study is clinical pathology application of GPC3 protein in PLC. Purified rabbit anti-human GPC3 polyclonal antibody was used to assess GPC3 expression in 276 paraffin-embedded tissues and tissue microarray from PLC. Immunohistochemical studies showed that GPC3 is expressed in 74.5% PLC (182 of 244), whereas it is not detectable in hepatocytes from normal liver around haemangioma, and different pathological types has remarkable difference in GPC3 positive rate (p>0.05). In HCC and ICC, the positive rates of GPC3 are 78.2% (176/225) and 31.6% (6/19), respectively, the expression and distribution among them are also different. Comparing with less positive cells and weak staining in ICC, almost all cancerous cells in HCC are positive and have strong staining. No remarkable difference of GPC3 positive rate (p>0.05) were found in HCC tissues between well differentiationed (I-II grade) and poor differentiationed (III-IV grade), ^ 3cm and >3cm in diameter, serum level of AFP>20ug/L and AFP ^ 20ug/L.However, in small HCC and I-II grade well differentiationed HCC tissues, GPC3 positive rate are 82.9%(63/76)> 75.4%(43/57), respectively, suggesting that GPC3 protein could be useful for early diagnosis.In addition, GPC3 positive rate in HCC tissues has no remarkable difference with that of in serum level of AFP (77.8% versus 76%) , but in HCC that of serum AFP^20ug/L, GPC3 positive rate in HCC tissues could be 80.0%(44/55), especially in small HCC with AFP^20ug/L,the positive rate also be 71.4%(10/14), all suggest that GPC3 protein distributes widely in HCC tissues, could be a useful immunohistochemacal marker for diagnosis and distinguish. We study 23 recurrent HCC patients, and found that GPC3 positive ratein primary cancerous liver tissues and recurrent cancerous liver tissues are 87.0% and 91.3%, respectively. The high rate of GPC3 protein in recurrent cancerous liver tissues indicate that their be some relationship between GPC3 and HCC recurrent, many work should be done to make clear it.The third part of our study is establishment of GPC3 detection method in the serum and clinical application. Rabbit anti-human GPC3 polyclonal antibody was biotinylated. Based on the high affinity between biotin and streptavidin, pairs of antibody sandwich ELISA method was set up to detect GPC3 in the serum, and according to the present actual conditions, we judged results by the way of determining the quality. Through above-mentioned method, 684 serums were detected. It showed that GPC3 positive rate in the groups of cirrhosis, PLC, recurrent PLC is higher than normal person, etc groups (P<0.05). In diseases that are in connection with liver, Hepatitis B —>ICC —^Cirrhosis —+HCC —recurrent PLC, GPC3 positive rate in serum appears by low —?High order. In 42 cases of cirrhosis, the appearing rate of unusual AFP and GPC3 in serum is 45.2% and 42.9% respectively. Especially in patients of whose serum AFP are greater than 400ug/L, 5 of 6 are GPC3 positive, putting forward a meaningful subject that will study in the future. By following up a large number of cirrhosis cases, uniting serum AFP and GPC3 unusual, is it possible to diagnosis for early liver cancer and how to define. In 162 PLC patients without treatment, the positive rate of GPC3 in serum is 41.4%, obviously higher than that of in the group of normal person (4.1%) and the group of hepatitis B (8.6%). It suggested that detecting GPC3 in serum is favorable to diagnosis of the liver cancer. The positive rates of GPC3 are 45.9% (61/133) and 20.9% (6/29) respectively in the group of HCC and ICC and have remarkable difference between them (PO.05), indicating that it will have certain meanings to distinguish different pathological types of PLC. What merits special attention is that in small HCC (s$3cm in diameter) cases, the positive rate of GPC3 is 36.0% (9/25), indicating that detecting GPC3 in serum is favorable to the early diagnosis of liver cancer. The positive rate of GPC3 is 41.9% (13/31) in HCC cases with AFP^20ug/L, which suggests that GPC3 can remedy with AFP, decrease leaking of liver cancerdiagnosis because AFP is negative. Moreover, GPC3 positive rate hit up to 61.1% in 36 cases, indicate that GPC3 might be relationship with recurrent PLC, which may regard GPC3 as one of tumour marks that indicate recurrent PLC. In our research, we can indicate recurrent PLC from 77.7% by using AFP alone up to 88.9% by uniting serum AFP and GPC3. In addition, 28.6% (10/35) of the cases that cancer of colon metastasis to liver has GPC3 in serum, whether can regard appearance of GPC3 as a sign of cancer of colon metastasis to liver, the answer to this question will be a ques with actual meanings. We have also studied HCC patient before and 2-16 day after operation, find that changes of GPC3 in serum show many kinds of situations. We can see that GPC3 in the serum will not have disappeared at once after a radical cure excision, which indicate that it need some time to clear away GPC3 in the blood. Whether these changes can use for valuing the effect of treatment as AFP, the answer to this question will wait for a long time following up. In the research of this part, we compare the results of immunohistichemical to that of GPC3 in the serum of 33 cases. It is found that 20 of 24 cases are immunohistichemical positive, among them 10 cases of serum GPC3 are positive, accounted for 50%, showing that the behavior of GPC3 in the serum is different in immunohistichemical positive cases, the way that GPC3 enters the blood await further clarifying. We have compared the testing results of serum AFP, GPC3, CA19-9, AFU of 162 liver cancers, and find that to all 162 liver cancer cases, the detecting positive rate to PLC by uniting AFP +GPC3 + CA19-9+ AFU four indexes and even by AFP +GPC3 + CA19-9 three indexes appear up to 87.7% and 86.4%, respectively, promoting 22.3% and 21.0% respectively than using AFP alone. But the differences are greater in different pathological types. In HCC, the positive rates of AFP and GPC3 are obviously higher than ICC, and CA19-9 positive rate in ICC is obviously higher than HCC. To ICC, by uniting AFP +GPC3 + CA19-9+ AFU four indexes can only promote the detecting positive rate to 3.5% than using CA19-9 alone which means it is no big significance to unite AFP +GPC3 + CA19-9+ AFU.But for the diagnosis to HCC, the detecting positive rate reachs to 89.8% by uniting AFP +GPC3+ AFU, promoting 13.1% than using AFP alone. In HCC that diameter <3cm and >3cm,...
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