【摘要】 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。更多还原

【Abstract】 AIM: To study the function of human TNF receptor-75 (hTR75) and the interaction between human TNF receptor-55 (hTR55) and hTR75 in hTNFa-induced cytotoxicity. METHODS: HEp-2 cells were transfected with bicistronic expression vector of hTR75 gene, and HEp-2-A75 cells with intrinsic hTR55 and overexpressed hTR75 were obtained. Two hTNFa muteins with exclusive specificity for hTR55 or hTR75 were constructed, expressed in high-levels in E coli , and then purified. hTNFa-induced cytotoxicity was determined by crystal violet colorimetric method. RESULTS: The expression of hTR75 in HEp-2 cells was demonstrated by RT-PCR and indirect ELISA, and was quantified by binding of [125I]hTNFa and Scatchard analysis. The overexpressed hTR75 could markedly increase the susceptibility of HEp-2 cells to hTNFa. CONCLUSION: hTR75 could not only partially mediate hTNFa-induced cytotoxicity independently but also fulfill an accessory role in enhancing or synergizing hTR55-mediated cytotoxicity. It played a dual function in hTNFa-induced cytotoxicity in HEp-2 cells.更多还原