肠道病毒71型感染的星形胶质细胞中EV713C活性与NF-κB信号转导效率的关联  

Correlation between EV-type 3C protease activity and NF-κB signal transduction efficiency in astrocytes infected with enterovirus 71

作　　者：魏容[1] 肖光军[1] 刘艳婷[1] 杨娜[1] WEI Rong;XIAO Guangjun;LIU Yanting;YANG Na(Department of Clinical Laboratory,Suining Central Hospital,Suining,Sichuan 629000,China)

机构地区：[1]遂宁市中心医院检验科,四川遂宁629000

出　　处：《安徽医药》2021年第10期2022-2027,共6页Anhui Medical and Pharmaceutical Journal

基　　金：遂宁市科技计划项目(2015s15)。

摘　　要：目的探究肠道病毒71型感染的星形胶质细胞中肠病毒71(EV71)3C蛋白酶活性与核因子κB(NF-κB)信号转导效率的关联。方法本研究起止时间为2018年3月至2019年3月。通过将EV71病毒感染星形胶质细胞后,经过质粒提取、转化、定点突变、基因转变等步骤,将TANK结合激酶1(TBK1)复合体切割后,分别导入肿瘤坏死因子受体相关因子2(TRAF2)质粒和TANK结合激酶1(TBK1)质粒,观察其对NF-kB表达的影响。同时,EV71感染后,通过RT-PCR和Western blotting法对细胞内相关基因表达,并对EV713C活性进行检测,得到蛋白酶活性与NF-kB表达关系,判断其关联。结果EV71感染可诱导TBK1和其复合体蛋白TAB 123的降解,并呈现时间依赖性(24h:TAB1:125.86±39.87;TAB2:352.12±42.36;TAB3:195.20±29.75),但对于肿瘤坏死因子受体相关因子2(TRAF2)和TANK结合激酶1(TBK1)表达无影响。同时,细胞内白细胞介素-6(IL-6)(19.4±0.62)、白细胞介素-8(IL-8)(13.8±2.89)、白细胞介素-12(IL-12)(13.0±0.68)及IL-1β(14.6±0.96)的表达均在12h后显著提高;TAB2可诱导NF-kB激活,而与3C共同表达时可显著抑制NF-kB表达,3C亦可显著抑制由TAK1复合体作用诱导NF-kB的激活。不同浓度EV71感染后,细胞内EV713C活性出现明显差异,而随着蛋白酶活性的增加,细胞内NF-kB mRNA和蛋白的表达均成明显的下调趋势。结论肠道病毒71型感染的星形胶质细胞中EV713C可通过调节TAK1蛋白表达,达到抑制NF-κB信号转导的作用。Objective To investigate the association between the activity of enterovirus 71 type 3C protease and NF-κB signal transduction efficiency in astrocytes infected with enterovirus 71.Methods The starting and ending time of this study was from March 2018 to March 2019.After infecting astrocytes with EV71 virus,the Tank-bound kinase 1(TBK1)complex was cleaved after plasmid extraction,transformation,site-directed mutagenesis and gene transformation,and then transferred into the tumor necrosis factor receptor-associated factor 2(TRAF2)plasmid and the TBK1 plasmid respectively to observe its effect on NF-κB expression.At the same time,after EV71 infection,the expression of intracellular related genes and EV713C protease activity were detected by RT-PCR and Western blotting,and the correlation was determined according to the relationship between activity and NF-κB expression.Results EV71 infection induced the degradation of TBK1 and its complex protein tab 1/2/3 in a time-dependent manner(24 h:tabl:125.86±39.87;TAB2:352.12±42.36;TAB3:195.20±75),but had no effect on the expression of TRAF2 and TBK1.At the same time,IL-6(19.44±0.62),IL-8(13.80±0.89),IL-12(13.02±0.68)and IL-1β(14.6±0.96)increased after 12 hours of infection;TAB2 could induce the activation of NF-κB,but co-expression with 3C could significantly inhibit the expression of NF-κB.3C could also significantly inhibit the activation of NF-κB induced by TAK1 complex.However,with the increase of protease activity,the expression of NF-κB mRNA and protein decreased significantly.Conclusion Enterovirus 71 type 3C protease in astrocytes infected with enterovirus 71 can inhibit the transcription of NF-κB by regulating the expression of TAK1 protein.

关 键 词：肠道病毒感染 星形细胞 EV713C蛋白 转化生长因子β活化激酶-1 核因子-ΚB信号通路 

分 类 号：R725.1[医药卫生—儿科]