0bJective :To construct 1he vector of dan inant negative mutant fom of Rab7 (Rab7T22N ) ,and analyze 1he effect of Rab7T22N overexpressbn on 1he production of cytokines in RAW 264.7 macrophages induced by poly I:C .M e1hods:The vector of Rab7T22N was obtained by PCR site-directed mutegenesis .The vectors of Rab7 and Rab7T22N were transient^ transfected hto RAW 264.7 eels by Ipo- son e .Rab7 protein expression was detected by 1he m eihod of W estem bbt .The production of FN-fS ,P10 ,TNF~a and H.fS were m easured after temsfected RAW 264 .7 eels w ere stm ulated by poly I :C for different tin e .R esulte :Can paied w ith 1he control cells temsfected w ith m ock vector, 1he protein expression of Rab7 were significant^ in proved in cell lines temsfected with Rab7 and Rab7T22N .Oveiexpression of Rab7 inhibited pofy I:C induced expression of M-P,P 10,TNF-a and L~I(3 in RAW 264.7 eels .Hew ever ,overexpression of Rab7T22N signifrantfy promoted 1he expression of 1he above cytokines in RAW 264 .7 macrophages induced by po]y I:C .Conclusion :Rab7 hhibits 1he expression of FN-fS ,P10 ,TNF~a and LH.(3 in po]y I:C stin ulated Raw264 .7 eels in a GTP-binding dependentm anner .The experin ents m ay lay a foundation for further study on the role of iab7 in TLR3 signaling paihways.%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.
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