肿瘤坏死因子受体Ⅱ196位点基因多态性与SLE相关性及功能研究

Association and function study of tumor necrosis factor receptorⅡ position 196 polymorphism with systemic lupus erythematosus

目的:调查中国汉族人群肿瘤坏死因子受体Ⅱ(TNFRⅡ)196位基因多态性与SLE的关系并构建野生和突变的逆转录病毒载体以研究其功能差异。方法:利用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)方法检测了106例SLE和119例健康人TNFRⅡ196位的基因型。扩增TNFRⅡ196McDNA克隆到PMD18-T载体上,定点突变为TNFRⅡ196R,然后亚克隆到逆转录病毒载体PLXSN上(PLXSN-TNFRⅡ196M和PLXSN-TNFRⅡ196R)并分别转染大鼠系膜细胞,以ELISA法观察对系膜细胞产生IL-6的影响。结果:(1)SLE组TNFRⅡ196R等位基因型明显高于正常组(35.2%vs14.3%,P<0.05);(2)成功构建野生型和突变型重组逆转录病毒载体;(3)rhTNFα刺激后196R型转染系膜细胞产生IL-6明显高于196M型(P<0.05)。结论:TNFRⅡ196R与中国汉族人群SLE相关,其可以高效转导TNFα的信号,可能参与SLE发病。

AIM： To investigate the assoeiation of gene polymorphism at position 196 of tumor necrosis factor receptor Ⅱ （TNFR Ⅱ ） with systemic lupus erythematosus （SLE） in Chinese, and establish recombinant retroviral vector to analyze the function of the TNFR Ⅱ 196M/R. METHODS： The genotype at position 196 of TNFR Ⅱ was determined by PCR- RFLP in 106 SLE patients and 119 healthy controls in china. Human TNFR Ⅱ 196M cDNA were amplified by PCR and cloned into PMD18 - T vector. Then, PMD18 - TNFRⅡ 196R was induced by site - directed mutagenesis. The recombinant T vector, PMD18 - TNFRⅡ196M and PMD18- TNFRⅡ 196R, were subcloned into retroviral vector PLXSN. Both normal and variant were transfected into rat mesangial cell. The effects of TNFα on production of sTNFRⅡ and IL - 6 were study by ELISA. RESULTS ： （ 1 ） The frequency of TNFR Ⅱ 196R allele was significantly higher than those in controls （35.2% vs 14.3%, P 〈 0.05）; （2） The recombinant retroviral vector （PLXSN- TNFR 196M and PLXSN-TNFR 196R） was constructed successfully; （3） rhTNFα caused a significant increase in IL- 6 production by rat mesangial cells transfected with PLXSN- TNFR Ⅱ 196R than that with TNFR Ⅱ 196M. CONCLUSION： These data indicate that TNFR Ⅱ 196R allele is associated with SLE in the Chinese. TNFR Ⅱ 196R transduces the signals of TNFα more effectively than TNFR Ⅱ 196M, which may be involved in the pathogenesis of SLE.