摘要:
Objective To investigate the effect of DDX60 on the replication of Japanese en-cephalitis virus(JEV),and preliminarily determine the mechanisms.Methods The mouse micro-glia C8-B4 cells were infected with JEV,with those infected with ultraviolet-inactivated JEV as con-trol.RT-qPCR was performed to detect the DDX60 expression levels in cells.Enzyme-linked immu-nosorbent assay(ELISA)was used to measure the levels of inflammatory cytokines.DDX60 knock-down plasmids or overexpressed DDX60 plasmids were transfected into C8-B4 cells using Lipo-fectamine 2000, and meanwhile, the cells transfected with empty plasmid served as the control group.RT-qPCR and Western blotting were applied to detect the expression levels of viral RNA and protein.The levels of inflammatory cytokines were assayed by ELISA.Results The DDX60 mRNA level was significantly lower in JEV group than in control group(P<0.05,P<0.01).The levels of viral RNA and protein were significantly decreased in DDX60 overexpression group as compared with control group(P<0.05,P<0.01),while those in DDX60 knock-down group were strikingly increased(P<0.01,P<0.001).Besides,the inflammatory cytokines levels were significantly in-creased in JEV group by contrast with inactivated JEV group(P <0.05, P <0.01, P <0.001) .The expression level of inflammatory cytokines was obviously reduced in DDX60 knock-down group compared with the control group(P<0.05),while that in DDX60 overexpression group was notably magnified(P<0.05).Conclusion JEV infection can significantly inhibit DDX60 expression in C8-B4 cells,and promote the release of inflammatory cytokines.DDX60 transfection prominently in-creases the levels of inflammatory cytokines in C8-B4 cells infected with JEV.In conclusion,DDX60 inhibits JEV replication by enhancing cellular immune response.%目的 探索DDX60对日本脑炎病毒(Japanese encephalitis virus, JEV)复制的影响,并初步确定其影响机制.方法 以小鼠小胶质C8-B4细胞作为研究对象,对其进行JEV感染实验,以感染紫外灭活JEV细胞作为对照组,采用RT-qPCR(实时定量逆转录聚合酶链反应)测定感染后两组细胞中DDX60的表达,采用酶联免疫吸附实验(ELISA)检测两组细胞中免疫因子的水平.利用Lipofectamine 2000将DDX60敲降或过表达质粒转染入C8-B4细胞,对照组细胞转染空载质粒,采用RT-qPCR和Western印迹检测病毒RNA和蛋白水平变化,采用ELISA检测细胞中免疫因子表达水平的变化.结果 JEV组细胞中DDX60 mR-NA表达量显著低于正常细胞组(P<0.05, P<0.01); DDX60过表达组中病毒RNA水平和蛋白水平明显降低(P<0.05, P<0.01),而DDX60敲减组中其水平显著升高(P<0.01, P<0.001);与JEV失活组相比,JEV组细胞中免疫因子的水平显著提高(P <0.05, P <0.01, P <0.001);与正常对照组相比, DDX60敲减组中细胞免疫因子的表达水平明显下降(P<0.05),而DDX60过表达组中免疫因子的表达水平明显上升(P<0.05).结论 JEV感染可显著抑制C8-B4细胞中DDX60的表达,并促进细胞中免疫因子的释放;DDX60可增强感染病毒的C8-B4细胞中免疫因子的表达.综上,DDX60通过提高细胞的免疫反应进而抑制病毒的复制.