JEV感染对小鼠睾丸间质细胞信号通路、炎症因子及睾酮分泌影响的研究  

Effects of JEV infection on signaling pathway,inflammatory factors and testosterone secretion in mouse testicular interstitial cells

作　　者：何松 陈阊峥 汤德元[1] 曾智勇[1] 王彬[1] 黄涛[1] 罗柳 周飘 毛茵茗 廖正波 陈旭 袁盛林 胡雯雯 周敏 HE Song;CHEN Chang-zheng;TANG De-yuan;ZENG Zhi-yong;WANG Bin;HUANG Tao;LUO Liu;ZHOU Piao;MAO Yin-ming;LIAO Zheng-bo;CHEN Xu;YUAN Sheng-lin;HU Wen-wen;ZHOU Min(College of Animal Science,Guizhou University,Guiyang,Guizhou 550025,China)

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025

出　　处：《中国预防兽医学报》2023年第12期1264-1271,共8页Chinese Journal of Preventive Veterinary Medicine

基　　金：贵州大学重点项目(黔科合平台人才[2018]5781-8)。

摘　　要：为探究日本乙型脑炎病毒(JEV)感染睾丸间质细胞(LC)后对其信号通路、炎症反应和睾酮分泌水平的影响,本研究以MOI 1的JEV感染小鼠LC(TM3细胞)后不同时间采用荧光定量PCR(qPCR)检测TM3细胞中Toll样受体3(TLR3)、维甲酸诱导基因-Ⅰ(RIG-I)、NF-κB及干扰素调节因子3(IRF3)信号通路相关蛋白基因mRNA的转录水平并确定JEV的最佳感染时间。结果显示,与空白对照组相比,JEV感染后12 h各蛋白基因mRNA的转录水平均显著升高(P<0.05),随着JEV感染时间的延长各蛋白基因mRNA转录水平逐渐降低至与空白对照组无显著差异,因此选择12 h作为后续JEV感染细胞的最佳时间。将JEV感染TM3细胞12 h后,采用western blot检测TM3细胞中TLR3、RIG-I、其下游信号通路相关蛋白NF-κB、IRF3及NF-κB和IRF3的磷酸化水平;采用间接免疫荧光试验(IFA)检测TM3细胞中NF-κB和IRF3蛋白的入核;通过间接ELISA检测TM3细胞中睾酮的分泌水平。Western blot结果显示,JEV感染TM3细胞后12 h TLR3、RIG-I、NF-κB、IRF3蛋白的表达及NF-κB、IRF3的磷酸化水平均显著升高(P<0.05)。IFA结果显示,空白对照组细胞核形态完整,绿色荧光均弥散分布于细胞和细胞质之中,而JEV感染后12 h出现CPE的TM3细胞核溶解和破碎,有些细胞核变形,绿色荧光出现在细胞核中,且荧光信号明显增强。ELISA结果显示,JEV感染后12 h TM3细胞上清中IL-6、IFN-β分泌水平极显著升高(P<0.01),睾酮分泌水平显著下降(P<0.05)。利用TLR3和RIG-I siRNA转染TM3细胞,以干扰TLR3和RIG-I的表达,采用western blot检测这两个siRNA分别对TLR3和RIG-I的干扰效果;干扰TLR3和RIG-I表达后以MOI 1的JEV感染TM3细胞,12 h后经ELISA检测细胞上清中IL-6、IFN-β和睾酮的分泌水平。SiRNA干扰试验结果显示,与空白对照细胞及转染siNC的细胞相比,目的基因siRNA转染的TM3细胞中TLR3和RIG-I蛋白的表达水平均极显著降低(P<0.01);ELISA结果显示,与JEV感染的�In order to investigate whether inflammation occurs,the signaling pathway and testosterone secretion levels after Japanese encephalitis virus(JEV)infects testicular interstitial cells(LC),in this study,the mRNA transcription level of the toll-like receptor 3(TLR3),retinoic acid-inducible gene-I(RIG-I),NF-κB and signal pathway related protein genes of interferon regulatory factor 3(IRF3)in mouse testicular interstitial cells(TM3 cells)was detected by fluorescence quantitative PCR(qPCR)at different time points after MOI 1 JEV infection,and the optimal infection time of JEV were determined.The results showed that compared with the blank control group,the mRNA transcription levels of all tested genes were significantly increased at 12 hpi(P<0.05),and the mRNA transcription levels of each gene gradually decreased to no significant difference with the blank control group in the extension of JEV infection.Therefore,12 hours was selected as the follow-up detection time of JEV-infected cells.Follow-up tests were performed at 12 hours after post JEV infection(hpi)on infected TM3 cells.Western blot was used to detect the expression level of TLR3,RIG-I,and its downstream signaling pathway-related proteins NF-κB,IRF3,as well as the phosphorylation of NF-κB and IRF3 in TM3 cells.In addition,the nucleation of NF-κB and IRF3 proteins in TM3 cells was detected by indirect immunofluorescence assay(IFA)and the level of testosterone secretion in TM3 cells was tested by indirect ELISA.Western blot results showed that the expression of TLR3,RIG-I,NF-κB and IRF3 protein and the phosphorylation of NF-κB and IRF3 were significantly increased in TM3 cells at 12 hpi(P<0.05).IFA results showed that the cell nuclei of the blank control group were intact,and green fluorescence was dispersed in the cells and cytoplasm.However,at 12 hours after JEV infection,nucleolysis and fragmentation occurred in the TM3 cells with CPE,green fluorescence appeared in the nucleus,and the fluorescence signal was significantly enhanced.ELISA results showed

关 键 词：日本乙型脑炎病毒 NF-κB TOLL样受体3 炎症因子 睾酮 

分 类 号：S852.65[农业科学—基础兽医学]