Development And Application Research Of Clinical Detection Reagents For Glypican-3 As An Early Diagnosis Marker For Hepatocellular Carcinoma

Background and PurposeCancer is a serious disease threat to human life, cancer mortality has been occupied to the first of human disease spectrum. Hepatocellular carcinoma (HCC) is the sixth largest tumor in the world. It is estimated that there were over one million new HCC patients worldwide each year, and about one million people died. China is one of the high incidence area in the world, the incidence rate is 20.37/10 million, and is the third higthest of common tumors. The treatment of HCC is still lack effective means, the mian treatment program is surgery in the early stag and the recurrence rate of surgery is higth. Tumor cells easily along the blood vessels and lymphatic for distant metastasis and HCC with poor prognosis.HCC early symptoms and signs are obvious or lack of specificity, so the rate of early diagnosisis low in HCC. It is often advanced when patients for medical treatment, so patients always lost access to treatment (Studies have shown that surgical treatment can be performed in early and mid-HCC, the 5-year survival rate is about 62.9% after surgical resection for small hepatocellular carcinoma and only 34.6% for large hepatocellular carcinoma).At the same time, due to HCC is lack sensitive to radiotherapy and chemotherapy, the prognosis is poor as a whole. The current treatment has no better strategy, so the effective way to improve survival, reduce mortality are effective screening in high-risk groups and the early diagnosis in HCC. Biomarker is most advantageous for early detection and diagnosis of small hepatocellular carcinoma. The most classic and recognized is alpha-fetoprotein (AFP) in the diagnosis of liver cancer, but positive detection rate is only 30 to 60%, and specificity is not enough. Therefore, to find new and effective diagnostic and therapeutic targets have become a key, difficult points and direction in the HCC study.Glypican-3 (GPC3) is a membrane heparan sulfate glycoprotein (heparan sulfate proteoglycan, HSPG). Its special structure affect a variety of biological effects of molecules, and play a key regulatory role in tissues, organs, and development. Since it was discovered in 1996, the relationship between glypican 3 and the tumor has become a hot research topic.1997, Hsu first reported the high expression of GPC3 mRNA in HCC, followed by more and more researchers confirmed in GPC3 both mRNA and protein were showed overexpressed in HCC, and this was occured in early stage of HCC.GPC3 positive expression rate was 81.5% in HCC tissue, while no expression in the cancer paraneoplastic and far HCC tissue in Miao Hui’s study.Di Tommaso’s studies have shown that, the sensitivity is 69% and a specificity of 91% in diagnosis of early-stage HCC by detecting GPC3. The level of serum GPC3 was increased in 53%(18/34) liver cancer patients, lower than the positive rate (72%) of HCC tissues, only 1 of the 20 patients with liver cirrhosis cases was high, normal controls and hepatitis were negative. In AFP negative HCC, GPC3 also has a higher expression rate. Ding, etc detected GPC3 mRNA in 41 AFP-negative HCC tissues and adjacent noncancerous tissues, in cancer tissue positive rate was 73.17%, adjacent tissues was only 9.76%, moreover GPC3 mRNA was weak expressed; the expression rate was 79.31% in large hepatocellular carcinoma (> 5 cm),41.67% in small HCC (≤5 cm),76.47% in poorly differentiated carcinoma (Ⅲ-Ⅳ class),42.86% in well-ifferentiated carcinoma (Ⅰ-Ⅱ level); mmunohistochemistry results showed that GPC3 was expressed in hepatoma cells, negative in normal liver cells, bile duct cells, endothelial cells, Ito cells and fibroblasts. There are no significant correlation between serum GPC3 and AFP levels, this is a certain extent compensate for the lack of AFP diagnosing for liver cancer.GPC3 expression status was also related to the prognosis of patients with hepatocellular carcinoma. In Shirakawa’s study,107 cases of hepatocellular carcinoma patients were divided into GPC3 positive and negative groups by immunohistochemical staining,5-yearfollow-up found that GPC3 positive group, mortality was significantly higher than the negative group (87.7% vs54.5%) In the 80 patients receiving surgical resection, there were no deaths (0/16) in GPC3-negative patients within five years.In summary, GPC3’s expression is closely related to the development and prognosis of hepatocellular carcinoma, and the combination of GPC3 plus AFP achieved a much improved sensitivtity compared with either GPC3 or AFP alone. So that GPC3 is consered as a great potential novel liver cancer marker. Because of its novelty, its clinical value and significance requires further evaluation and research. For its innovative, there is no detection reagent products can be large-scale clinical application. Therefore, research and development of high-end GPC3 immunological detection reagents is very necessary and has broad application prospects.Cytokeratin 19 (CK19), bile duct cell-specific keratin intermediate filaments, no expression in normal human liver cells. During embryonic development, embryonic stem cell express CK8,18,19, with the differentiation of the liver, normal liver cells do not express CK19, only expressed in bile duct cells. When liver cells become cancerous, and some cancer cells re-express CK19, as a part of the complete molecular structure, CK19 expression was negatively correlated with the degree of cell differentiation. When cell necrosis, CK19 released to serum as dissolve fragments form,and can be detected in the blood, detect indicators was CYFRA21-1. Clinical pathological specimen studies have showed that the degree of differentiation was worse, invasiveness is stronger, the degree of malignancy was higher when the CK19 was expressed in liver cancer. When highly metastatic human hepatoma cell line was inoculated in nude mice, it was also found CK19 levels in serum increased rise significantly with tumor progression. Li Yan detected 101 normal serum, the results was the normal reference value of the detection system. The serum of CK19 was higher than the maximum reference value in 22% of 108 patients with HCC, CK19 elevated and AFP normal in 11% of HCC patients. Nagai et al have reported that there were 33 cases (47.1%)with hinger elevated blood CK19 in 70 cases of liver cancer patients; 12.3% with CK19 higher than normal valual but AFP normal. The simultaneous detection of CK19 and AFP may have a certain degree of complementarity in diagnosis.Philosophy of transformation medical is the bridge between the basic medicine and clinical medicine effective fast bridge. How will the research of previous large-scale gene functional genomics, proteomics and functional proteomics applied to clinical research and diagnosis and treatment of disease as soon as possible,is the problem the biological medicine and clinical inspection and other fields think and attention and expectations implemented to solve. In this paper, we selected phosphatidylinositol phosphate proteoglycan-3(Glypican-3, GPC3), a great potential and novel tumor marker, used an advanced new immunological detection technology--chemiluminescence immune analysis method to carry out high-end immune quantitative assay development, and at the same time the immunohistochemistry to detect GPC3expression in hepatocellular carcinoma tissues characteristics was established. Combined detect serum GPC3,AFP, CK19 and do clinical diagnosis value of applied research, to solve the early diagnosis of hepatocellular carcinoma and prognosis monitoring urgent clinical need to tackle the practical problems.Methods: 1.Preparation of GPC3 antigen:GPC3 N-end was cloned into the prokaryotic expression vector PHUE, recombinant proteins was successful expression,and a large numberecombinant proteins were inducible expressed in E. coli. Recombinant protein was purified by affinity chromatography, to have activity target protein by dialysis and refolding and identificatied by Werstern-Blot.2. Preparation of anti-GPC3 monoclonal antibody:purified refolded recombinant protein as antigen to immunize BALB/c mice, when the titer was up to 1:50,000, using the classic method to prepare monoclonal antibodies, and the monoclonal antibodies were identified by Western-Blot and immunofluorescence.3. Paired antibodies screening:use a double-antibody sandwich method of time-resolved fluorescence immunoassay technology to filter out the highest fluorescence value of 2 monoclonal antibodies by f 2and 2 pairing from the prepared anti-GPC3 monoclonal antibodes, this two were large antibodies were numerously and prepared and purified.4. Development of GPC3 chemiluminescence immunoassay test kits:use an direct chemiluminescence immunoassay with double antibody sandwich method to develop the GPC3 clinical detection reagent, and evaluate the performance of the reagents, including:the drawing of the working curve, sensitivity tests, interferencetest, the temperature destructive experimental and clinical specimens.5. Development of GPC3 immunohistochemistry reagents:development of immunohistochemistry reagents, repair conditions, the blocking solution, and the concentration of first anti-concentration a secondary antibody were optimizated, and clinical specimens were tested.6. The detection and analysis of clinical specimens:detect GPC3 and CK19 in the serum of liver cancer patients, and associated with serum AFP and content analysis.Results:N-terminal gene of GPC3 was cloned into PHUE prokaryotic expression plasmid successfully and the PHUE/GPC3 prokaryotic expression plasmid was constructed successfully by PCR, restriction enzyme digestion and sequencing identified. GPC3 fusion protein was expressed in E. coli, the molecular weight of about 22 KDa and basically in line with the theoretical value. The expression products were mainly expressed in the form of inclusion body. After Recombinant protein inclusion body was washed and purited by Ni affinity chromatography, the purity was more than 90%. Specific band was at 22 KDa by Western-blot analysis with anti-HIS tag antibody, and this showed that this protein is the indeed fusion protein expression products. Purified protein was injected into mice, aftere five immune, the ffect price was up to 1:50,000, and then prepared by cell fusion,13 anti-GPC3 monoclonal antibodies were prepared and identified by Western-blot analysis, immunofluorescence.Results showed that monoclonal antibodies-selfmade and natural protein wre able to react specifically, so the preparation of monoclonal antibodies wre specific monoclonal antibodies. Coefficient of variation in the matched pair of antibodies,7D11 and 8G6, and this pair of antibodies to prepar GPC3 chemical luminescence immunoassay reagents, the GPC3-CLIA was good specific and with a wide linear range; analysis using time-resolved screening 2.8%-7.0% coefficient of variation analysis between 3.3%-6.1%; Aftern reagents placed at 37℃ for 7 days or 4℃ for six months, fluorescence was no indecreased and the CV<10% of the analysis, analysis of the inter-CV<10%,with a better stability. In serum of hepatocellular carcinoma GPC3 detection rate was 54.2% by using homemade kit. The United GPC3 and AFP in hepatocellular carcinoma detection rate was 80.7%, jointed CK19 and AFP HCC detection rate was 71.8%, AFP, GPC3 and CK19 combined detection rate was upgraded to 90.6%. Immunohistochemical reagents were developed by using the home-made antibody; detection rat was 72.5% in the development of hepatocellular carcinoma tisse.Conclusion:These results suggest that in this study PHUE/GPC3 prokaryotic expression plasmid was successfully constructed, and immune activity the recombinant protein was expressed in E. coli; 13 anti-GPC3 monoclonal antibody hybridoma cell lines wre screened and established, screening from these 13 Mabs to get a matched pair of antibodies, using this pair of antibody to establish GPC3 -ELCA reagents and all indicators (sensitivity, precision, specificity, stability, accuracy) reached the clinical testing requirements; using 7D11 to develop immunohistochemical chemical reagents, and the detection rate of hepatocellular carcinoma is consistent with literature reports. These clinical reagents laid a foundation for the differential diagnosis of hepatocellular carcinoma. Next, further optimization of the detection reagent will be made and more clinical data will be added to go on this research. We expect to push the achievement to the clinical application and industrialization. The implementation of the project is the application of translational medicine philosophy, and both give services to the human health industry and improve medical standards, at the same time the biomedical industry can also be driven, considerable economic benefits, with the important social and economic significance.