Rab7 GTPase负向调节巨噬细胞中poly I:C诱导的细胞因子的表达

Rab7 GTPase negatively regulate cytokines expression in macrophages induced by poly I:C

目的:通过构建Rab7的失活突变载体tRab7(Rab7T22N),研究Rab7失活突变后对poly I:C诱导的RAW264.7巨噬细胞中细胞因子的影响。方法:利用PCR定点突变法构建Rab7的失活突变载体Rab7T22N,将Rab7的真核表达载体及其突变体tRab7通过脂质体法瞬时转染RAW264.7细胞,通过Western blot方法检测Rab7的表达情况。poly I:C刺激转染细胞不同时间后检测细胞因子IFN-β、IP10、TNF-α和IL-1β的表达变化。结果:Western blot检测结果显示,与转染空质粒的对照细胞相比,转染Rab7和tRab7的细胞中Rab7的蛋白水平显著增高。Rab7过表达后,抑制了巨噬细胞RAW264.7中poly I:C刺激后IFN-β、IP10、TNF-α和IL-1β的产生,而Rab7失活突变体tRab7(Rab7T22N)过表达后显著促进了IFN-β、IP10、TNF-α和IL-1β的表达。结论:Rab7抑制了poly I:C刺激的巨噬细胞中IFN-β、IP10、TNF-α和IL-1β的表达,该抑制作用依赖于其GTP结合活性。本研究为进一步阐明Rab7在TLR3信号转导通路中的作用奠定了基础。

Objective：To construct the vector of dominant negative mutant form of Rab7（Rab7T22N）,and analyze the effect of Rab7T22N overexpression on the production of cytokines in RAW264.7 macrophages induced by poly I：C.Methods：The vector of Rab7T22N was obtained by PCR site-directed mutagenesis.The vectors of Rab7 and Rab7T22N were transiently transfected into RAW264.7 cells by liposome.Rab7 protein expression was detected by the method of Western blot.The production of IFN-β,IP10,TNF-α and IL-1β were measured after transfected RAW264.7 cells were stimulated by poly I：C for different time.Results：Compared with the control cells transfected with mock vector,the protein expression of Rab7 were significantly improved in cell lines transfected with Rab7 and Rab7T22N.Overexpression of Rab7 inhibited poly I：C induced expression of IFN-β,IP10,TNF-α and IL-1β in RAW264.7 cells.However,overexpression of Rab7T22N significantly promoted the expression of the above cytokines in RAW264.7 macrophages induced by poly I：C.Conclusion：Rab7 inhibits the expression of IFN-β,IP10,TNF-α and IL-1β in poly I：C stimulated Raw264.7 cells in a GTP-binding dependent manner.The experiments may lay a foundation for further study on the role of rab7 in TLR3 signaling pathways.