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Discovery of New Sites in Fibronectin Ⅲ5 Associated with Hepatocellular Carcinoma Using Antibody Technology

Author: LiuRong
Tutor: SunQiHong
School: PLA Military Academy of Medical Sciences
Course: Immunology
Keywords: Primary liver cancer Tumor markers Fibronectin
CLC: R735.7
Type: Master's thesis
Year: 2008
Downloads: 33
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Abstract

[Objective] primary liver cancer (primary carcinoma of liver, PCL) is 6th in the world, China's three most common malignancies, including primary hepatocellular carcinoma (hepatocellular carcinoma, HCC) and primary bile duct cancer are two main types, the incidence of the former accounted for more than 90% of the overall incidence of PCL. According to the International Cancer Research Center estimates that the global liver cancer in 2000 a total of 56.4 million people, more than half of them (about 55%) occurred in China, the number of liver cancer in China was 30.6 million, while liver cancer deaths and up to 300,000, and there is still an upward trend in recent years of primary liver cancer morbidity and mortality. The treatment of liver cancer is still surgical resection is the preferred, early removal is the key to improve the survival rate of patients; smaller the tumor, the higher five-year survival rate. But the onset of primary liver cancer often more hidden, early HCC is difficult to find, once the symptoms, most to advanced. Due to the use of liver cancer markers AFP, liver cancer early diagnosis and early radical surgical treatment possible, but its sensitivity is difficult to meet clinical needs, more ideal tumor marker remains to be further research and exploration. The purpose of this study is the use of the characteristics of the antibody technology and secreted proteins explore new tumor markers for primary liver cancer. [Method] In this study, a highly metastatic hepatoma cell line HCCLM-6 cells total protein immune BALB / c mice from mouse spleen cells with SP2 / 0 fusion establishment of hybridoma cell lines, cells of HCCLM-6 serum the culture supernatant and total cellular protein as a combined screening antigen, the filter cell secretory protein antibody and hepatoma cells by the ELISA method. By immunohistochemical screening for liver cancer tissue-specific antibodies, and were identified by indirect immunofluorescence method of in different metastatic liver cancer cell lines (highly metastatic cell lines HCCLM-6 and low metastatic cell lines HCC97-L No metastatic cell lines HepG2), the distribution of normal liver cell line L-02 and metastatic lung giant cell carcinoma cell line 95D; selected by screening Uni-ZAP XR adult liver cDNA expression library with hepatoma-specific a monoclonal antibody to the antigen recognized identification, in order to find more desirable and effective diagnostic marker of primary liver cancer. [Results] by cell fusion experiments to establish the mouse anti HCCLM-6 hybridoma cell lines 28. 28 hepatoma-specific monoclonal antibodies were identified by indirect enzyme-linked immunosorbent assay (ELISA) and immunohistochemical staining and indirect immunofluorescence method. ELISA detection results suggest QGA062, QEC104 QAG013 QAC081 by QFH307 and QAG063 these six strains of monoclonal antibodies to the protein in serum-free culture supernatant of hepatoma cell lines HCCLM-6 total protein and HCCLM-6 cells were significantly higher than normal liver cell line L-02 and the mouse myeloma cell line SP2 / 0. Immunohistochemistry and indirect immunofluorescence identification results suggest that five monoclonal antibodies that QAG013 QGA062 QAC081 QAE018, and QFH307 can specifically recognize the protein in HCC tissues and different hepatoma cell line, and in the normal liver tissue and normal liver cell line L-02, there is no obvious expression. Combined indirect enzyme-linked immunosorbent assay, immunohistochemistry and indirect immunofluorescence identification results, the selection of monoclonal antibodies QAG013, QGA062, QAC081, QAE018 and QFH307 adult liver cDNA expression library screening, preliminary identification QAG013 QGA062 QAC081 and QFH307 these four strains of monoclonal antibodies recognize an antigen fibronectin (fibronectin, FN). To QAG013, QGA062, QAC081 after QFH307 positive clones shear induced by plasmid expression product as an antigen, each of these four monoclonal antibodies identified by Western blot, sequencing results are positive clone insert the length of the fragment and the insertion position of the monoclonal The location of the antigen recognized by the antibody were analyzed, the results suggest that monoclonal antibodies, respectively, recognize the FN molecule, three different epitopes, especially QGA062 mAb epitope identified only within a range of 16 amino acids (YTVSLVAIKGNQESPK). Found both in this paragraph amino acid sequence homology comparison of the human and mouse differ only 4,8 and 13 amino acids, the peptide antigen epitope analysis of the results showed a higher score analysis area is located in the peptide carboxyl end of the segment, suggesting that the first 13 amino acids is likely to play a key role in the composition of the QGA062 epitope peptides found to further study the relationship between FN and cell adhesion, angiogenesis and tumor FN new tumor sites laid the foundation. [Conclusion] The study, prepared by hybridoma technique mouse anti-human highly metastatic hepatoma cell line HCCLM-6 monoclonal antibody 28 by indirect enzyme-linked immunosorbent assay, immunohistochemistry and indirect immunofluorescence method to determine which 5 monoclonal antibody QAG013 QGA062 QAC081 QAE018 and QFH307 in HCC tissues and hepatoma cell line with high specificity. Adult liver cDNA expression library screening results suggest that monoclonal antibodies QAG013, QGA062, QAC081 and antigen recognition QFH307 as FN, respectively, for three different epitopes in the FN molecule, especially QGA062 mAb epitope recognition only within the range of the 16 amino acids (YTVSLVAIKGNQESPK), laid the foundation for further study of the relationship between the FN and cell adhesion, angiogenesis and tumor and FN new tumor sites.

Discovery of New Sites in Fibronectin Ⅲ5 Associated with Hepatocellular Carcinoma Using Antibody Technology

Abstract

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