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目的:研究人肿瘤坏死因子受体75(hTR75)在hTNFα所引发的细胞毒效应中的功能。方法:用电脉冲将hTR75基因表达载体转入只有内源性hTR55表达的HEp-2细胞中构建了两种TNF受体同时表达的HEp-2-A75细胞株。通过定点突变构建及在大肠杆菌中高效表达,分离纯化出两种分别对hTR55以及hTR75受体具有选择结合活性的hTNFα突变体。野生型hTNFα及其这两种突变体对HEp-2以及HEp-2-A75细胞的细胞毒效应采用比色法测定并进行了比较。结果:RT-PCR以及间接ELISA的结果表明hTR75在RNA转录以及蛋白质翻译水平均获得了表达,其表达量由Scatchard分析确定。HEp-2细胞中hTR75的存在可以明显增强HEp-2细胞对hTNFα的敏感性。结论:hTR75受体在介导hTNFα的细胞毒活性中具有双重功能,即hTR75不但自身可介导部份hTNFα所引发的细胞毒效应,它对hTR55所介导的细胞毒效应还具有相当的协同作用。
AIM: To investigate the role of human tumor necrosis factor receptor 75 (hTR75) in the cytotoxic effect induced by hTNFα. METHODS: HEp-2-A75 cell line was constructed by electropulsing the hTR75 gene expression vector into HEp-2 cells with endogenous hTR55 expression only. Through site-directed mutagenesis and high expression in E. coli, two hTNFα mutants with selective binding activity to hTR55 and hTR75 respectively were isolated and purified. The cytotoxic effects of wild type hTNF [alpha] and both of these mutants on HEp-2 and HEp-2-A75 cells were measured and compared by colorimetry. Results: The results of RT-PCR and indirect ELISA showed that hTR75 was expressed in RNA transcripts and protein translation levels, and its expression was determined by Scatchard analysis. The presence of hTR75 in HEp-2 cells can significantly enhance the sensitivity of HEp-2 cells to hTNFα. CONCLUSION: The hTR75 receptor has a dual function in mediating the cytotoxic activity of hTNFα. In other words, hTR75 not only mediates the cytotoxic effect induced by hTNFα, but also has considerable synergism with the cytotoxic effect mediated by hTR55 effect.