DEN2感染对MyD88沉默的小鼠树突状细胞相关膜分子表达及炎症因子分泌的影响

The effect of expressing related membrane molecule and secreted inflammation factor on MyD88 silenced murine dendritic cells infected with DEN2

目的:探讨髓样分化因子88(MyD88)在DEN2感染BMDC后对其成熟及相关炎症因子分泌的影响。方法:利用rmIL-4、rmGM-CSF联合诱导培养BMDC;针对BMDC MyD88基因,设计并合成3对MyD88 siRNA,脂质体法转染BMDC;通过Western blot筛选一对高沉默效率的MyD88 siRNA;常规方法增殖及鉴定DEN2;直接免疫荧光及RT-PCR鉴定DEN2吸附BMDC;流式细胞术分析对照组、感染组、RNAi组、感染RNAi组、无义RNAi组BMDC膜表面分子CD86、MHCⅡ的表达,双抗体夹心ELISA法检测上清液中IP-10、TNF-α及IFN-α的分泌水平。结果:经细胞因子诱导培养后可获得CD11c表达为70.85%±2.66%的相对未成熟的BMDC;与空白对照组相比,序列1～3组的MyD88蛋白质表达率分别降低28.36%±14.26%、54.20%±22.93%、73.14%±11.80%,其中序列3的沉默效率最高,具有显著统计学意义(P<0.01);DEN2可吸附BMDC。与对照组比较,DEN2感染组DC膜表面分子CD86表达降低,MHCⅡ增高,感染RNAi组的MHCⅡ、CD86表达无明显变化;与对照组比较,DEN2感染组分泌TNF-α、IFN-α及IP-10的水平均增高,而感染RNAi组分泌的IP-10、IFN-α、TNF-α则无明显变化。结论:DEN2可吸附BMDC,并促使其成熟及细胞因子分泌;siRNA可沉默MyD88,有效阻断DEN2感染BMDC的胞内信号传导及其所引起的成熟及炎症因子分泌。

Objective：To observe the effects of Myeloid Differentiation Factor 88（MyD88） on the maturity and secretion of cytokines in Bone Marrow-derived Dendritic Cells（BMDC） infected with DEN2.Methods： BMDC were generated with stimulation of rmIL-4,rmGM-CSF.Three pairs of MyD88 siRNA were designed,and were transfected with lipofection.The expression of MYB88 was detected with Western blot,and one of the highest silence effective siRNA was selected.DEN2 virues were proliferated and identified by conventional methods.The adherence of DEN2 to BMDC was observed by Direct Immunofluorescence and RT-PCR.The expression of membrane molecule CD86,MHCⅡ were detected by FCM,in control group,infected group,RNAi group,infected RNAi group,and no sense RNAi group.And then,IP-10,TNF-α,and IFN-α were detected by ELISA.Results：The expression reached at 70.85%±2.66% of CD11c in relative immature BMDC was acquired with stimulation of cytokine;compared with the control group,the expression of MyD88 in Sequence 1-3 group reduced 28.36%±14.26%,54.20%±22.93%,73.14%±11.80%.The highest silence efficiency was Sequence 3（P0.01）;BMDC be able to adhered for DEN2.Compared with control group,the expression of CD86 reduced,but MHCⅡ increased in infected group,and the levels of CD86 and MHCⅡ no changed in infected RNAi group.Compared with control group,the secretion of TNF-α,IFN-α,and IP-10 increased in infected group,but no changed in infected RNAi group.Conclusion：The maturity and secretion of cytokines of BMDC were adhered for DEN2;MyD88 were silenced by siRNA,which can effectively blocked intracellular signal and that interrupted the maturity and secretion of cytokines of BMDC were infected DEN2.