Recostruction Of SEA-B7.1 Double Signals On Human Hepatocellular Carcinoma Cells And Analysis Of Its Immunological Effect

Human hepatocellular carcinoma (HCC) cells do not induce an effective immunological rejection process because of their weak antigenicity, abnormal expression of MHC molecules and deletion or deficiency of costimulatory molecules. To solve this problem, we transduced the genes of staphylococcal enterotoxin A (SEA) and B7.1 into a HCC cell line HHCC, to make the cells to express the molecules of SEA and B7.1. The purpose is to improve the capability of HCC remarkably of inducing immunological rejection. In our study we ligated the genes of B7.1 and SEA with a special linker and analyzed the flexibility, antigenicity, Hoop&Woods hydrophilicity and episode of ' recombinant toxin CD80-Linker-SEA in order to keep each molecule's the biological activity at its most. Then we constructed retroviral vectors containing the genes of SEA, B7.1and B7.1-linker-SEA and introduced them into packaging cell line PA317 through liposome-mediated transfection. Under selection with G418 the G418-resistant colonies that can produce virus were isolated, and the high-titer producers were identified using NIH3T3 cells. HHCC cells were infected with the high-titer retroviral supernatant and selected under G418. For the first time the HCC cell line HHCC-PS11and HHCCBS which expressed SEA protein and B7.1-linker-SEA fused protein respectively were obtained. The cytotoxicity assay proved that the expression of SEA led to an improved activation of anti-tumor lymphocytes. The Vmax(Maxim rate of lysis ) of HHCC-PS11 and HHCCBS by T lymphocytes were 1.72×10~4/ml·hr and 1.67× 10~4/ml·hr which were higher than that of HHCC(1.19× 10~4/mlhr).Transduced HCC cells were analyzed for HLA expression, and it was found that a majority of the cells expressed HLA-1 molecules but no HLA- DR molecules. After blocking the HLA- I molecules by HLA- I mAb, the cytotoxicity of T...