Stable Transfection And Expression Of TNF-α Gene And Its Mutants In Human Cell Line MG63

Tumor necrosis factor-alpha (TNF-α), produced chiefly by activated monocytes and macrophages, is a potent proinflammatory cytokine and plays critical role in immune response and inflamemation. It regulates cell proliferation, differentiation and apoptosis. It exists as two forms, the 26kD membrane TNF-α(mTNF-α) and the 17kD soluble TNF-α(sTNF-α),which is derived from proteolytic cleavage of the 26kD membrane form. The two forms of TNF-αelicit a particularly broad biological activity via binding to its receptors, TNFR1 and TNFR2.But some differences are present between these two forms of TNF-αin their biological effects, modes and mechanisms of action. And more, the binding of mTNF-αto TNFR resulted in not only the transduction of a signal from TNFR to target cell (positive signaling) but also the transmission of a signal from mTNF-α, like a receptor, to effector cell (reverse signaling).We had constructed three kinds of retroviral vectors, pLS-TNF, pLTM-TNF, pLWt-TNF. The pLS-TNF (a secreted TNF-αmutant in which the TNF-αsignal peptide was replaced by IL-2 signal peptide) expressed secreted 17-kDa TNF-αonly. The pLTM-TNF (a membrane TNF-αmutant in which the zymolytic site capable of transforming mTNF-αinto sTNF-αhas been deleted) expressed only membrane bound 26-kD TNF-α, which could not be secreted by the cell. The pLWt-TNF expressed the wild type TNF-α. The retrovirus packaging cell line PA317 was respectively transfected with pLS-TNF, pLTM-TNF and pLWt-TNF with the mediation of lipofectin. Cell lines PA/S-TNF, PA/TM-TNF and PA/Wt-TNF secreting different retrovirus were obtained.In this study, we chosed the osteosarcoma cell line MG63 expressing low level of TNF-α. We respectively transfected three types of TNF-αgenes into the MG63 cell line by the three kinds of retroviral vectors with TNF-αgenes. And then establishd the cell lines respectively expressing three types of TNF-αgenes, which lays the foundation for further studying the biological effects of TNF-αand the TNF-αsignaling pathway.1. The expression of TNF-αin MG63 cell lineThe results from RT-PCR were showed that MG63 cells transcribed low level of TNF-αmRNA. The results from western blotting were also showed that MG63 cells expressed low level of TNF-αprotein. And more, the results of FCM and ELISA were consistent with that above.In a word, these dates above are consistent with others reported that MG63 cell is the cell line expressing littlee TNF-α.2. Establishment of cell with expression fo different forms of TNF-αMG63 were transfected respectively with three types of high titer retrovirus containing different TNF-αgenes. As shown by indirect immunofluorescence technique, FCM, ELISA and Western blotting, the different TNF-αgenes were effectively expressed by MG63. The results showed that both cells and supernatants of MG63/wTNF contained high level of TNF-α. The cells of MG63/mTNF and the supernatants of cultured MG63/sTNF cells contained high level of TNF-α.3. Investigation of the biological function in vitroWe chosed the L929 cell line as the target cell, which is sensitive to human TNF-α. The results showed that the supernatant of culture cells expressing sTNF-αwas cytotoxic to L929 cell line. The fixed cells expressing mTNF-αwas cytotoxic to L929 cell line. While both fixed cells and supernatants of cultured cells expressing wTNF-αshowed cytotoxity. Furthermore, these cytotoxic effects could be blocked by anti-TNF-αantibodies.4. The duration of the three cell lines expressing TNF-αgeneThe aim of this study was to establish the cell lines expressing three types of TNF-αgenes with stability, so we observed the duration of the cell line expressing TNF-αgene by FCM. The results from FCM were showed that, after the cell line being cultured several generations, the level of TNF-αwas obviously reduced. But the cell line was cloned once more, and the cell lines expressing high level of target gene were obtained.Conclusion: All the datas above suggested: the cell lines expressing three types of TNF-αgenes were successfully established, which could express the genes with stability and efficiency. The genes expressed all showed their cytotoxic effect.