Experimental Study On B7-1 Gene Transfection Of Hepatocellular Carcinoma Cells

Objective The immune response to solid tumour mainly depend on active T cells. T cell activation requires a signal delivered via interaction of the TCR with specific Ag on MHC molecules and a costimulatory signal. The most fully characterized costimulatory signal is mediated by the binding of CD80 and CD86 on APC to their receptor CD28 on T cells. In the absence of costimulation signal, T cell receptors Ag interaction appear to induce an anergic state or apoptosis in T cells.Hepatocellular carcinoma is believed to be a poorly immunogenic tumour. Its cells expressed costimulatory molecules B7-land B7-2 so low levels that insufficient costimulation fail to induce an effective anti-tumour immune response. We tried to establish an HCC cell line that strongly expressed B7-1 by transfecting human B7-1 gene into human HCC cells and evaluated the role of B7-1 expression in the induction of lymphocytes and primary cytolytic activity against HHCC.Methods We investigated the expression of B7-1 in hepG2 cell by flow cytometric (FCM) analysis and reverse transcription-polymerase chain reaction(RT-PCR). Retroviral transduction was applied in transfecting PLXSN-B7-1 into packaging cell line, NIH3T3, and high-titer clone was acquired, then virus stock were used to infect the hepG2 cell. Meanwhile, growth curve were determinated by cytometry. After transfected with B7-1 gene. The lymphocytes of heathy subject were co-cultured with hepG2 and hepG2/hB7-1vaccines, lymphocyte proliferation was evaluated by MTT assay.Results FCM revealed that hepG2 cells expressed B7-1 on the cell surface, however, the expression levels of B7-1 were very low (positive rate: 3.66%). RT-PCR showed that hepG2 cells were negative for B7-1 at the messenger RNA(mRNA) level. The both results were in close agreement.The clone strongly expressing B7-l(positive rate: 92.2% ) was picked out. After transfected with B7-1 gene, the tumour cell proliferation was lagged.The results revealed hepG2/hB7-l cells stimulated vigorous proliferation of lymphocytes, much higher than that of hepG2 vaccines. Finally, we observed the effects on changes in cytotoxic activity of induced CTL. The result showed the cytotoxicity was reinforced, cytotoxic activity of CTL against hepG2/hB7-l cell were 5.86, 1.63, 1.43 times that much of hepG2 groups at E/T 10:1, 25:1, 100:1 respectively (p<0.01).Conclusion The results suggested that the transfection of B7-1 gene could established B7-1 high-expressing positive clone in hepG2 cell, transfection with B7-1 gene can inhibit the proliferation of hepG2 cells and stimulate vigorous proliferation of lymphocytes to reinforce cytotoxic activity of induced CTL in vitro.