SLE 相关基因 IFIT1 免疫调节功能的初步研究.docVIP

SLE 相关基因 IFIT1 免疫调节功能的初步研究 【摘要】 目的：研究系统性红斑狼疮（SLE）相关基因 IFIT1 的功能。方法：首先将 IFIT1 从穿梭表达克隆转入真核表达克隆，然后转导进入 Jurkat 细胞系，用有限稀释法筛选单克隆；再用 CD3 和 CD28 刺激单克隆，检测膜分子的表达、细胞凋亡、增生、细胞因子分泌等免疫生物学指标；最后应用 RNAi 技术，下调 IFIT1 的表达，检测细胞因子分泌，比较分析 IFIT1 的功能。结果：IFIT1 的过度表达在 T 细胞中能诱导白细胞介素 IL-4 和 IL-10 的分泌增加，下调 IFIT1 的转录水平后能部分下调 IL-4 和 IL-10 表达，而与细胞膜分子的表达、细胞的增生、凋亡无关。结论：SLE 相关新基因 IFIT1 在细胞因子的产生中起了重要作用，可能与 Thl/Th2 的失衡有关。 【关键词】 系统性红斑狼疮；IFIT1；细胞因子；基因干预 〔Abstract〕Objective To study the function of IFIT1, a novel gene associated with the systemic lupus erythematosus (SLE). MethodsFirst an expression clone of IFIT1 was generated by performing an LR recombination reaction between the entry clone from Genecopoiea and a GatewayTM destination vector. Then the vector was transfected into the Jurkat T cells. Single clone cells expressing EGFP and IFIT1-EGFP were selected by standard limiting dilution method. The stable transfectants of EGFP and IFIT1-EGFP were stimulated with anti-CD3 and anti-CD28. Cell surface antigen expression were detected by flow cytometry. The apoptosis and expansion of each transfectant were detected according to the manufacture’s instruction. The culture supematants were assayed for cytokines by sandwich ELISA. Furthermore transfection of gene silence vector which could produce anti-IFIT1-specifics small RNA duplexes partially blocked the expression of overexpressed IFIT1 in Jurkat T cel1. The same assays were done by stimulation with anti-CD3 and anti-CD28. ResultsThere were no significant difference in the expression level of the various cell surface molecules, apoptosis and expansion among Jurkat, Jurkat/EGFP, and Jurkat/IFIT1-EGFP transfectants. But it was found that the Jurkat/IFIT1-EGFP could enhance the production of IL-4 and IL-10. After knocking down the expression of IFIT1 by RNAi, the levels of IL-4 and IL-10 production were partially decreased.ConclusionThe results indicate the SLE related-gene IFIT1 may play an important role in the production of cytokines and may int