JAK/STAT和PI3K/AKT信号通路介导小反刍兽疫病毒宿主细胞天然免疫的研究

Study on Innate Immune Response of Peste Des Petits Ruminants Virus Infected Cells Mediated by JAK/STAT and PI3K/AKT Signaling Pathways

为研究JAK/STAT和PI3K/AKT信号通路在小反刍兽疫病毒(PPRV)宿主细胞天然免疫中的作用,在PPRV感染山羊肾细胞24、48和72 h后,分别用MTT试验和间接免疫荧光试验(IFA)检测病毒感染细胞活力及其在细胞中的分布;用qRT-PCR和Western-blot分别检测病毒蛋白、Nectin-4受体、JAK/STAT和PI3K/AKT信号通路及其下游信号分子表达水平的变化。结果表明,PPRV感染24、48和72 h后的细胞存活率分别为99.53%、77.12%和66.87%,病毒H和N蛋白表达水平显著增加(P<0.05);但Nectin-4表达无显著变化(P>0.05);p-STAT1/STAT1比值极显著升高(P<0.01);ISG20、ISG15、IRF9、IRF3、IFNβ和IFNα表达水平显著或极显著升高(P<0.05或P<0.01);p-AKT表达水平极显著升高(P<0.01);p-AKT/AKT、p-NFκB/NFκB、p-GSK/GSK和p-CREB/CREB比值均显著或极显著升高(P<0.05或P<0.01)。综上,PPRV感染能激活JAK/STAT和PI3K/AKT信号通路,使细胞处于抗病毒状态。

To further investigate the effects of JAK/STAT and PI3K/AKT signaling pathways on the innate immune reponse of the PPRV-infected cells,goat kidney cells(LDG-2)were cultured and infected with PPRV for 24,48 and 72 h.Subsequently,the cell viability was detected by MTT assay,and the propagation and distribution of the virus in host cells was observed by indirect immunofluorescence assay(IFA)at different time points above.The expression levels of the viral proteins H and N,cellular recepotor Nectin-4,JAK/STAT and PI3K/AKT signaling pathways and downstream signaling molecules,were determined by qRT-PCR and Western-blot.The results showed that the survival rate of the cells infected by PPRV for 24,48 and 72 h was 99.53%,77.12%and 66.87%,respectively,and the expression levels of viral H and N proteins were significantly increased(P<0.05),but no significant changes of the expression level of cellular receptor Nectin-4 were observed(P>0.05).Moreover,the ratio of p-STAT1/STAT1 and the expression levels of downstream signaling molecules of JAK/STAT pathway such as ISG20,ISG15,IRF9,IRF3,IFNβand IFNαwere significantly increased(P<0.05 or P<0.01).The expression level of p-AKT and the ratios of p-AKT/AKT,p-NFκB/NFκB,p-GSK/GSK and p-CREB/CREB were significantly higher than those of the cell controls(P<0.05 or P<0.01).In conclusion,both of the JAK/STAT and PI3K/AKT signaling pathways were activated due to PPRV infection,which led to the antiviral status of host cells.