本文主要研究内容
作者申周（2019）在《TRIM21对A549细胞中呼吸道合胞病毒（RSV）复制的影响》一文中研究指出：目的:呼吸道合胞病毒(Respiratory Syncytial Virus,RSV)是一种有包膜的、单负链RNA病毒,属副粘病毒科。RSV可引起严重的急性下呼吸道感染,是导致小儿病毒性肺炎最常见的病原体之一。三结构域(Tripartite motif,TRIM)蛋白家族隶属于E3泛素连接酶家族,家族中多数成员的氨基末端具有典型的RBCC结构域。辅以羧基末端PRY、SPRY、ARF等特殊结构域,使TRIM蛋白家族在生物学功能上具有多样性。TRIM蛋白家族在细胞凋亡、自噬、癌症发生中均扮演重要的角色,近年来,越来越多的研究显示:TRIM蛋白家族还在固有免疫,尤其是抗病毒固有免疫应答调控中发挥重要作用。许多TRIM蛋白通过诱导Ⅰ型干扰素,发挥抗病毒作用。然而,并非所有的TRIM家族成员与入侵宿主的病毒“不共戴天”,极少数TRIM家族成员如TRIM6、TRIM23、TRIM29等可以负向调控抗病毒免疫应答而发挥促病毒复制的作用。TRIM蛋白在RSV病毒感染中发挥何种作用,尚不清楚。课题组前期通过基因表达谱芯片技术,获得了感染RSV的A549细胞的基因表达谱数据,筛选到五种可能与RSV感染密切相关的TRIM蛋白,包括TRIM21,TRIM22,TRIM14,TRIM25和TRIM38。本研究主要锁定TRIM21蛋白,欲探讨其在RSV感染中的作用,这将有助于人们深入理解宿主和病毒的相互作用,为阐明RSV致病机制,开发新的靶向TRIM家族蛋白的药物奠定基础。方法:1明确RSV感染对A549细胞TRIM21蛋白表达的影响。1.1 GFP-RSV以不同感染复数(MOI)感染A549细胞,通过荧光显微镜观察GFP-RSV融合病毒其标签蛋白GFP在A549细胞中的表达;qPCR检测不同MOI RSV感染对TRIM21 mRNA表达的影响,以确定RSV感染的最佳MOI。1.2以最佳MOI的RSV感染A549细胞,在不同时间点,采用qPCR和Western blot方法分别检测TRIM21 mRNA和蛋白表达水平的变化,明确RSV感染对A549细胞TRIM21蛋白表达的影响,并为后续试验确定检测的最佳时间点。2构建pCAGGS-HA-TRIM21真核表达质粒和pGL4.10-IFNβpromoter荧光素酶报告质粒。3应用多种手段明确TRIM21的亚细胞定位。3.1 RSV感染A549细胞48h后,分离核蛋白和浆蛋白,用Western blot检测TRIM21蛋白在细胞核和细胞浆蛋白中的表达,明确TRIM21蛋白在细胞中的定位。3.2将pCAGGS-HA-TRIM21真核表达质粒转染爬片生长的A549细胞24h后,再感染RSV 24h,TRIM21蛋白经免疫荧光染色后,于正置荧光显微镜下观察其表达定位。4明确TRIM21对RSV病毒复制的影响。4.1应用siRNA敲低A549细胞TRIM21基因的表达后,再感染RSV,于感染后0h、24h、48h收集细胞,提取细胞总RNA和蛋白,应用qPCR和Western blot方法验证TRIM21的敲低效果。4.2应用siRNA敲低A549细胞TRIM21基因的表达后,再感染RSV,于感染后24h和48h,采用qPCR和病毒滴定的方法分别检测RSV N基因mRNA表达水平及病毒滴度的变化,探讨敲降TRIM21对RSV病毒复制的影响。4.3用pCAGGS-HA-TRIM21真核表达质粒,转染A549细胞,应用qPCR和Western blot方法分别检测TRIM21 mRNA和蛋白表达水平,验证其过表达效果。4.4将pCAGGS-HA-TRIM21真核表达质粒转染A549细胞24h后,再感染RSV,采用qPCR和病毒滴定的方法分别检测RSV N基因mRNA表达水平以及病毒滴度的变化,探讨过表达TRIM21对RSV病毒复制的影响。5明确TRIM21蛋白对RSV诱导的Ⅰ型干扰素的影响。5.1应用siRNA敲低A549细胞TRIM21基因的表达后,再感染RSV 24h,qPCR检测Ⅰ型干扰素及下游一系列干扰素刺激基因(ISGs)的mRNA表达变化,探讨敲降TRIM21对RSV诱导的Ⅰ型干扰素及ISGs基因表达的影响。5.2将siTRIM21、pGL4.10-IFNβpromoter荧光素酶报告质粒和海肾荧光素酶参照质粒pGL4.10-TK共转染A549细胞24h后,应用双荧光素酶报告实验检测干扰素β启动子活性,明确敲降TRIM21对IFNβ基因启动子活性的影响。5.3将pCAGGS-HA-TRIM21真核表达质粒转染A549细胞24h后,再感染RSV 24h,qPCR检测Ⅰ型干扰素及下游一系列ISGs基因的mRNA水平,探讨过表达TRIM21对RSV诱导的Ⅰ型干扰素及ISGs基因表达的影响。5.4将pCAGGS-HA-TRIM21真核表达质粒、pGL4.10-IFNβpromoter荧光素酶报告质粒和海肾荧光素酶参照质粒pGL4.10-TK共转染A549细胞24h后,应用双荧光素酶报告实验检测干扰素β启动子活性,明确过表达TRIM21对IFNβ基因启动子活性的影响。6明确TRIM21和Ⅰ型干扰素之间的相互调控作用。6.1用不同浓度梯度的人源IFNβ重组蛋白刺激A549细胞12h,qPCR检测TRIM21基因的表达情况;以最优剂量的人源IFNβ重组蛋白刺激A549细胞,于不同时间点收集细胞,提取总RNA,qPCR检测TRIM21基因表达水平。明确Ⅰ型干扰素是否可以诱导TRIM21蛋白的表达,即明确TRIM21是否作为一种ISGs基因受Ⅰ型干扰素调控。6.2将pCAGGS-HA-TRIM21真核表达质粒转染A549细胞24h后,qPCR检测IFNβ基因的表达水平,明确过表达TRIM21对IFNβ基因表达的影响;将pCAGGS-HA-TRIM21真核表达质粒、pGL4.10-IFNβpromoter荧光素酶报告质粒和海肾荧光素酶参照质粒pGL4.10-TK共转染A549细胞24h后,应用双荧光素酶报告实验检测干扰素β启动子活性,明确过表达TRIM21对IFNβ基因启动子活性的影响。结合两个实验结果,明确TRIM21是否参与调控细胞的Ⅰ型干扰素通路。7明确TRIM21可通过干扰素非依赖机制促进RSV病毒复制。应用干扰素基因自然缺失的Vero细胞,探讨TRIM21是否通过干扰素依赖机制促进RSV病毒复制。7.1应用siRNA敲低Vero细胞TRIM21基因的表达后,再感染RSV,采用Western blot和病毒滴定的方法分别检测RSV病毒蛋白表达及病毒滴度的变化,探讨敲降TRIM21对Vero细胞中RSV病毒复制的影响。7.2将pCAGGS-HA-TRIM21真核表达质粒转染Vero细胞24h后,再感染RSV,采用Western blot和病毒滴定的方法分别检测RSV病毒蛋白表达及病毒滴度的变化,探讨过表达TRIM21对Vero细胞中RSV病毒复制的影响。8明确TRIM21可通过自噬机制促进RSV病毒复制。8.1以最佳MOI的RSV感染A549细胞后,在不同时间点收集细胞并提取细胞总蛋白,采用Western blot方法检测LC3Ⅱ蛋白的表达,验证RSV感染是否可诱导A549细胞的自噬。8.2应用siRNA敲低A549细胞TRIM21基因的表达后,再感染RSV,采用Western blot方法检测A549细胞LC3Ⅱ蛋白的表达,探讨敲降TRIM21对自噬水平的影响。8.3将pCAGGS-HA-TRIM21真核表达质粒转染A549细胞24h后,再感染RSV,采用Western blot方法检测A549细胞LC3Ⅱ蛋白的表达,探讨过表达TRIM21对自噬水平的影响。8.4应用siRNA敲低A549细胞TRIM21基因的表达后,用自噬诱导剂(雷帕霉素Rapamycin,100nM)处理A549细胞以回复自噬水平,再感染RSV。感染后24h通过倒置荧光显微镜观察GFP-RSV病毒荧光;用qPCR检测RSV N、RSV F、RSV NS1基因mRNA表达水平。以验证自噬诱导剂是否可以回复因敲低TRIM21而引起的病毒复制减少。8.5将pCAGGS-HA-TRIM21真核表达质粒转染A549细胞24h后,用自噬抑制剂(渥曼青霉素Wortmannin,100nM)处理A549细胞以抑制自噬水平,再感染RSV。感染后24h通过倒置荧光显微镜观察GFP-RSV病毒荧光;用qPCR检测RSV N、RSV F、RSV NS1基因mRNA表达水平。以验证自噬抑制剂是否可以回复因过表达TRIM21而引起的病毒复制增加。结果:1 RSV感染对A549细胞TRIM21蛋白表达的影响。1.1用倒置荧光显微镜初步观察GFP-RSV融合病毒其标签蛋白GFP在A549细胞中的表达。感染RSV 24h后,随着感染复数的增加,镜下荧光斑点数量和亮度有明显增加。qPCR结果显示,当MOI=1时,RSV对TRIM21的诱导作用最强,则将其确定为最佳感染复数。1.2.RSV(MOI=1)感染A549细胞,在感染后0h、6h、12h、24h、36h、48h,qPCR结果显示:感染后12h,TRIM21 mRNA的表达开始有明显上调,持续升高,直至检测时间点48h;Western Blot结果表明,感染后12h,TRIM21蛋白的表达开始有明显上调,持续升高,直至检测时间点48h。即:RSV感染可诱导TRIM21蛋白高表达,呈明显的时间依赖关系。2成功构建pCAGGS-HA-TRIM21真核表达质粒和pGL4.10-IFNβpromoter荧光素酶报告质粒2.1成功构建pCAGGS-HA-TRIM21载体,经酶切和测序鉴定正确。2.2成功构建pGL4.10-IFNβpromoter荧光素酶报告基因载体,经酶切和测序鉴定正确。3利用多种手段确定TRIM21的亚细胞定位3.1 RSV感染A549细胞48h后,分离核蛋白和浆蛋白,Western blot检测结果显示,TRIM21蛋白大多数表达在细胞浆之中。3.2用pCAGGS-HA-TRIM21真核表达质粒转染爬片生长的A549细胞24h后,再感染RSV 24h,免疫荧光染色,荧光显微镜观察结果显示,TRIM21蛋白几乎全部分布于细胞浆之中,呈片状散在分布。4 TRIM21蛋白对RSV病毒复制的影响4.1将TRIM21的siRNA转染A549细胞24h后,再感染RSV,在感染后0h,24h,48h收集细胞,分别进行qPCR和Western Blot检测。结果显示,与对照组相比,敲低TRIM21后,TRIM21 mRNA及蛋白表达均显著下降,提示敲低效果较好。4.2将siTRIM21转染A549细胞24h后,再感染RSV,于感染后24h和48h,收集细胞,进行qPCR和病毒滴度检测。qPCR结果显示,与对照组相比,敲低TRIM21后,RSV N基因mRNA表达有明显下降;病毒滴定(感染后24h)结果显示,与对照组相比,敲低TRIM21可明显降低RSV病毒滴度。4.3将pCAGGS-HA-TRIM21真核表达质粒转染A549细胞,收集细胞,分别进行qPCR和Western blot检测。结果显示,与pCAGGS-HA空载转染组相比,pCAGGS-HA-TRIM21转染组中TRIM21mRNA及蛋白的表达均有显著升高,提示过表达效果较好。4.4将pCAGGS-HA-TRIM21真核表达质粒转染A549细胞24h后,再感染RSV 24h,收集细胞,进行qPCR和病毒滴定检测。qPCR结果显示,与对照组相比,过表达TRIM21可明显提高RSV N基因mRNA的表达;病毒滴定结果显示:过表达TRIM21可明显提高病毒滴度。5 TRIM21蛋白对RSV诱导的Ⅰ型干扰素的影响5.1用siRNA敲低A549细胞TRIM21基因的表达后,再感染RSV,感染后24h,收集细胞,进行qPCR,结果显示,敲降TRIM21可明显降低Ⅰ型干扰素及其下游一系列干扰素刺激基因(ISGs)的mRNA的表达。5.2将siTRIM21、pGL4.10-IFNβpromoter荧光素酶报告质粒和海肾荧光素酶参照质粒pGL4.10-TK共转染A549细胞24h后,再感染RSV,感染后24h,进行双荧光素酶报告实验。结果显示,敲降TRIM21可明显降低干扰素β启动子活性。5.3将pCAGGS-HA-TRIM21真核表达质粒转染A549细胞24h后,再感染RSV 24h,收集细胞,进行qPCR检测。结果显示,过表达TRIM21可明显上调Ⅰ型干扰素及下游一系列ISGs基因的mRNA的表达。5.4将pCAGGS-HA-TRIM21真核表达质粒、pGL4.10-IFNβpromoter荧光素酶报告质粒和海肾荧光素酶参照质粒pGL4.10-TK共转染A549细胞24h后,再感染RSV 24h,进行双荧光素酶报告实验。检测结果显示,过表达TRIM21可明显增加干扰素β启动子活性。6 TRIM21与Ⅰ型干扰素的相互诱导作用6.1用不同浓度梯度的人源IFNβ重组蛋白刺激A549细胞12h,qPCR结果显示,以5ng/ml极低浓度的人源IFNβ重组蛋白就足以诱导TRIM21基因的高表达,不同剂量对TRIM21的诱导作用差别不大。确定最优剂量后,以该剂量的人源IFNβ重组蛋白刺激A549细胞,分别于0h,4h,8h,12h和16h收集细胞,进行qPCR检测。结果显示,人源IFNβ重组蛋白刺激A549细胞后4h,即可明显提高TRIM21的表达水平,于12h达到峰值。6.2将pCAGGS-HA-TRIM21真核表达质粒转染A549细胞24h后,收集细胞,进行qPCR检测。结果显示:过表达TRIM21可明显提高IFNβ基因mRNA的表达水平;将pCAGGS-HA-TRIM21真核表达质粒、pGL4.10-IFNβpromoter荧光素酶报告质粒和海肾荧光素酶参照质粒pGL4.10-TK共转染A549细胞24h后,进行双荧光素酶报告基因检测。结果显示,过表达TRIM21可明显提高干扰素β启动子活性。综合两个实验结果,提示:TRIM21参与调控A549细胞的Ⅰ型干扰素通路。7 TRIM21可通过干扰素非依赖机制促进病毒复制7.1应用siRNA敲低干扰素基因自然缺失的Vero细胞的TRIM21基因表达之后,再感染RSV 24h,进行Western blot和病毒滴定。Western blot结果显示:敲低TRIM21明显降低RSV病毒蛋白表达;病毒滴定(感染后24h)结果显示:敲低TRIM21可明显降低RSV病毒滴度。7.2用pCAGGS-HA-TRIM21真核表达质粒转染Vero细胞24h后,再感染RSV 24h,收集细胞,进行Western blot和病毒滴定。Western blot结果显示:过表达TRIM21可明显升高RSV病毒蛋白表达;病毒滴定(感染后24h)结果显示:过表达TRIM21可明显提高RSV病毒滴度。8 TRIM21可通过自噬机制促进RSV病毒复制。8.1以最佳MOI的RSV感染A549细胞后,于0h、24h、48h收集细胞,提取细胞总蛋白,进行Western blot。Western blot结果显示,RSV感染细胞后,LC3Ⅱ蛋白表达增高,即RSV成功诱导了A549细胞的自噬,一直持续到检测点48h。8.2应用siRNA敲低A549细胞TRIM21基因的表达后,再感染RSV,于感染后24h,进行Western blot。结果显示,无论是否有RSV感染,敲低TRIM21后均能降低LC3Ⅱ蛋白表达,提示敲低TRIM21表达可以降低细胞自噬水平。8.3将pCAGGS-HA-TRIM21真核表达质粒转染A549细胞24h后,再感染RSV,于感染后24h,进行Western blot。结果显示,无论是否有RSV感染,过表达TRIM21后均能提高LC3Ⅱ蛋白表达,提示过表达TRIM21可以提高细胞自噬水平。8.4应用siRNA敲低A549细胞TRIM21基因的表达后,用自噬诱导剂处理A549细胞以回复自噬水平,再感染RSV。感染后24h用倒置荧光显微镜初步观察,敲低TRIM21之后,与对照组相比,GFP-RSV病毒荧光斑点数量明显减少,与之前结果一致。在敲低TRIM21的基础上,再加入自噬诱导剂,GFP-RSV病毒荧光斑点数量明显增加;qPCR结果显示,自噬诱导剂不会影响TRIM21基因的表达,但与单独敲低TRIM21组比较,自噬诱导剂可以明显回复RSV N、RSV F、RSV NS1基因的表达水平。提示:自噬诱导剂可以回复因敲低TRIM21引起的病毒复制减少。8.5将pCAGGS-HA-TRIM21真核表达质粒转染A549细胞24h后,用自噬抑制剂处理A549细胞以抑制自噬水平,再感染RSV。感染后24h用倒置荧光显微镜初步观察,过表达TRIM21之后,与对照组相比,GFP-RSV病毒荧光斑点数量明显增加,与之前结果一致。在过表达TRIM21的基础上,再加入自噬抑制剂,GFP-RSV病毒荧光斑点数量明显减少;qPCR结果显示,自噬抑制剂不会影响TRIM21基因的表达,但与单独过表达TRIM21组比较,自噬抑制剂可以明显抑制RSV N、RSV F、RSV NS1基因的表达水平。提示:自噬抑制剂可以抑制因过表达TRIM21引起的病毒复制增加。结论:1.RSV感染可以上调A549细胞中TRIM21 mRNA及蛋白的表达。2.TRIM21蛋白主要定位于细胞浆中。3.TRIM21蛋白可以促进RSV复制。4.TRIM21与Ⅰ型干扰素可以相互诱导,并很可能存在一个正反馈环路。5.TRIM21蛋白可通过干扰素非依赖机制促进RSV复制。6.TRIM21蛋白可通过自噬机制促进RSV复制。
Abstract
mu de :hu xi dao ge bao bing du (Respiratory Syncytial Virus,RSV)shi yi chong you bao mo de 、chan fu lian RNAbing du ,shu fu nian bing du ke 。RSVke yin qi yan chong de ji xing xia hu xi dao gan ran ,shi dao zhi xiao er bing du xing fei yan zui chang jian de bing yuan ti zhi yi 。san jie gou yu (Tripartite motif,TRIM)dan bai jia zu li shu yu E3fan su lian jie mei jia zu ,jia zu zhong duo shu cheng yuan de an ji mo duan ju you dian xing de RBCCjie gou yu 。fu yi suo ji mo duan PRY、SPRY、ARFdeng te shu jie gou yu ,shi TRIMdan bai jia zu zai sheng wu xue gong neng shang ju you duo yang xing 。TRIMdan bai jia zu zai xi bao diao wang 、zi shi 、ai zheng fa sheng zhong jun ban yan chong yao de jiao se ,jin nian lai ,yue lai yue duo de yan jiu xian shi :TRIMdan bai jia zu hai zai gu you mian yi ,you ji shi kang bing du gu you mian yi ying da diao kong zhong fa hui chong yao zuo yong 。hu duo TRIMdan bai tong guo you dao Ⅰxing gan rao su ,fa hui kang bing du zuo yong 。ran er ,bing fei suo you de TRIMjia zu cheng yuan yu ru qin su zhu de bing du “bu gong dai tian ”,ji shao shu TRIMjia zu cheng yuan ru TRIM6、TRIM23、TRIM29deng ke yi fu xiang diao kong kang bing du mian yi ying da er fa hui cu bing du fu zhi de zuo yong 。TRIMdan bai zai RSVbing du gan ran zhong fa hui he chong zuo yong ,shang bu qing chu 。ke ti zu qian ji tong guo ji yin biao da pu xin pian ji shu ,huo de le gan ran RSVde A549xi bao de ji yin biao da pu shu ju ,shai shua dao wu chong ke neng yu RSVgan ran mi qie xiang guan de TRIMdan bai ,bao gua TRIM21,TRIM22,TRIM14,TRIM25he TRIM38。ben yan jiu zhu yao suo ding TRIM21dan bai ,yu tan tao ji zai RSVgan ran zhong de zuo yong ,zhe jiang you zhu yu ren men shen ru li jie su zhu he bing du de xiang hu zuo yong ,wei chan ming RSVzhi bing ji zhi ,kai fa xin de ba xiang TRIMjia zu dan bai de yao wu dian ding ji chu 。fang fa :1ming que RSVgan ran dui A549xi bao TRIM21dan bai biao da de ying xiang 。1.1 GFP-RSVyi bu tong gan ran fu shu (MOI)gan ran A549xi bao ,tong guo ying guang xian wei jing guan cha GFP-RSVrong ge bing du ji biao qian dan bai GFPzai A549xi bao zhong de biao da ;qPCRjian ce bu tong MOI RSVgan ran dui TRIM21 mRNAbiao da de ying xiang ,yi que ding RSVgan ran de zui jia MOI。1.2yi zui jia MOIde RSVgan ran A549xi bao ,zai bu tong shi jian dian ,cai yong qPCRhe Western blotfang fa fen bie jian ce TRIM21 mRNAhe dan bai biao da shui ping de bian hua ,ming que RSVgan ran dui A549xi bao TRIM21dan bai biao da de ying xiang ,bing wei hou xu shi yan que ding jian ce de zui jia shi jian dian 。2gou jian pCAGGS-HA-TRIM21zhen he biao da zhi li he pGL4.10-IFNβpromoterying guang su mei bao gao zhi li 。3ying yong duo chong shou duan ming que TRIM21de ya xi bao ding wei 。3.1 RSVgan ran A549xi bao 48hhou ,fen li he dan bai he jiang dan bai ,yong Western blotjian ce TRIM21dan bai zai xi bao he he xi bao jiang dan bai zhong de biao da ,ming que TRIM21dan bai zai xi bao zhong de ding wei 。3.2jiang pCAGGS-HA-TRIM21zhen he biao da zhi li zhuai ran pa pian sheng chang de A549xi bao 24hhou ,zai gan ran RSV 24h,TRIM21dan bai jing mian yi ying guang ran se hou ,yu zheng zhi ying guang xian wei jing xia guan cha ji biao da ding wei 。4ming que TRIM21dui RSVbing du fu zhi de ying xiang 。4.1ying yong siRNAqiao di A549xi bao TRIM21ji yin de biao da hou ,zai gan ran RSV,yu gan ran hou 0h、24h、48hshou ji xi bao ,di qu xi bao zong RNAhe dan bai ,ying yong qPCRhe Western blotfang fa yan zheng TRIM21de qiao di xiao guo 。4.2ying yong siRNAqiao di A549xi bao TRIM21ji yin de biao da hou ,zai gan ran RSV,yu gan ran hou 24hhe 48h,cai yong qPCRhe bing du di ding de fang fa fen bie jian ce RSV Nji yin mRNAbiao da shui ping ji bing du di du de bian hua ,tan tao qiao jiang TRIM21dui RSVbing du fu zhi de ying xiang 。4.3yong pCAGGS-HA-TRIM21zhen he biao da zhi li ,zhuai ran A549xi bao ,ying yong qPCRhe Western blotfang fa fen bie jian ce TRIM21 mRNAhe dan bai biao da shui ping ,yan zheng ji guo biao da xiao guo 。4.4jiang pCAGGS-HA-TRIM21zhen he biao da zhi li zhuai ran A549xi bao 24hhou ,zai gan ran RSV,cai yong qPCRhe bing du di ding de fang fa fen bie jian ce RSV Nji yin mRNAbiao da shui ping yi ji bing du di du de bian hua ,tan tao guo biao da TRIM21dui RSVbing du fu zhi de ying xiang 。5ming que TRIM21dan bai dui RSVyou dao de Ⅰxing gan rao su de ying xiang 。5.1ying yong siRNAqiao di A549xi bao TRIM21ji yin de biao da hou ,zai gan ran RSV 24h,qPCRjian ce Ⅰxing gan rao su ji xia you yi ji lie gan rao su ci ji ji yin (ISGs)de mRNAbiao da bian hua ,tan tao qiao jiang TRIM21dui RSVyou dao de Ⅰxing gan rao su ji ISGsji yin biao da de ying xiang 。5.2jiang siTRIM21、pGL4.10-IFNβpromoterying guang su mei bao gao zhi li he hai shen ying guang su mei can zhao zhi li pGL4.10-TKgong zhuai ran A549xi bao 24hhou ,ying yong shuang ying guang su mei bao gao shi yan jian ce gan rao su βqi dong zi huo xing ,ming que qiao jiang TRIM21dui IFNβji yin qi dong zi huo xing de ying xiang 。5.3jiang pCAGGS-HA-TRIM21zhen he biao da zhi li zhuai ran A549xi bao 24hhou ,zai gan ran RSV 24h,qPCRjian ce Ⅰxing gan rao su ji xia you yi ji lie ISGsji yin de mRNAshui ping ,tan tao guo biao da TRIM21dui RSVyou dao de Ⅰxing gan rao su ji ISGsji yin biao da de ying xiang 。5.4jiang pCAGGS-HA-TRIM21zhen he biao da zhi li 、pGL4.10-IFNβpromoterying guang su mei bao gao zhi li he hai shen ying guang su mei can zhao zhi li pGL4.10-TKgong zhuai ran A549xi bao 24hhou ,ying yong shuang ying guang su mei bao gao shi yan jian ce gan rao su βqi dong zi huo xing ,ming que guo biao da TRIM21dui IFNβji yin qi dong zi huo xing de ying xiang 。6ming que TRIM21he Ⅰxing gan rao su zhi jian de xiang hu diao kong zuo yong 。6.1yong bu tong nong du ti du de ren yuan IFNβchong zu dan bai ci ji A549xi bao 12h,qPCRjian ce TRIM21ji yin de biao da qing kuang ;yi zui you ji liang de ren yuan IFNβchong zu dan bai ci ji A549xi bao ,yu bu tong shi jian dian shou ji xi bao ,di qu zong RNA,qPCRjian ce TRIM21ji yin biao da shui ping 。ming que Ⅰxing gan rao su shi fou ke yi you dao TRIM21dan bai de biao da ,ji ming que TRIM21shi fou zuo wei yi chong ISGsji yin shou Ⅰxing gan rao su diao kong 。6.2jiang pCAGGS-HA-TRIM21zhen he biao da zhi li zhuai ran A549xi bao 24hhou ,qPCRjian ce IFNβji yin de biao da shui ping ,ming que guo biao da TRIM21dui IFNβji yin biao da de ying xiang ;jiang pCAGGS-HA-TRIM21zhen he biao da zhi li 、pGL4.10-IFNβpromoterying guang su mei bao gao zhi li he hai shen ying guang su mei can zhao zhi li pGL4.10-TKgong zhuai ran A549xi bao 24hhou ,ying yong shuang ying guang su mei bao gao shi yan jian ce gan rao su βqi dong zi huo xing ,ming que guo biao da TRIM21dui IFNβji yin qi dong zi huo xing de ying xiang 。jie ge liang ge shi yan jie guo ,ming que TRIM21shi fou can yu diao kong xi bao de Ⅰxing gan rao su tong lu 。7ming que TRIM21ke tong guo gan rao su fei yi lai ji zhi cu jin RSVbing du fu zhi 。ying yong gan rao su ji yin zi ran que shi de Veroxi bao ,tan tao TRIM21shi fou tong guo gan rao su yi lai ji zhi cu jin RSVbing du fu zhi 。7.1ying yong siRNAqiao di Veroxi bao TRIM21ji yin de biao da hou ,zai gan ran RSV,cai yong Western blothe bing du di ding de fang fa fen bie jian ce RSVbing du dan bai biao da ji bing du di du de bian hua ,tan tao qiao jiang TRIM21dui Veroxi bao zhong RSVbing du fu zhi de ying xiang 。7.2jiang pCAGGS-HA-TRIM21zhen he biao da zhi li zhuai ran Veroxi bao 24hhou ,zai gan ran RSV,cai yong Western blothe bing du di ding de fang fa fen bie jian ce RSVbing du dan bai biao da ji bing du di du de bian hua ,tan tao guo biao da TRIM21dui Veroxi bao zhong RSVbing du fu zhi de ying xiang 。8ming que TRIM21ke tong guo zi shi ji zhi cu jin RSVbing du fu zhi 。8.1yi zui jia MOIde RSVgan ran A549xi bao hou ,zai bu tong shi jian dian shou ji xi bao bing di qu xi bao zong dan bai ,cai yong Western blotfang fa jian ce LC3Ⅱdan bai de biao da ,yan zheng RSVgan ran shi fou ke you dao A549xi bao de zi shi 。8.2ying yong siRNAqiao di A549xi bao TRIM21ji yin de biao da hou ,zai gan ran RSV,cai yong Western blotfang fa jian ce A549xi bao LC3Ⅱdan bai de biao da ,tan tao qiao jiang TRIM21dui zi shi shui ping de ying xiang 。8.3jiang pCAGGS-HA-TRIM21zhen he biao da zhi li zhuai ran A549xi bao 24hhou ,zai gan ran RSV,cai yong Western blotfang fa jian ce A549xi bao LC3Ⅱdan bai de biao da ,tan tao guo biao da TRIM21dui zi shi shui ping de ying xiang 。8.4ying yong siRNAqiao di A549xi bao TRIM21ji yin de biao da hou ,yong zi shi you dao ji (lei pa mei su Rapamycin,100nM)chu li A549xi bao yi hui fu zi shi shui ping ,zai gan ran RSV。gan ran hou 24htong guo dao zhi ying guang xian wei jing guan cha GFP-RSVbing du ying guang ;yong qPCRjian ce RSV N、RSV F、RSV NS1ji yin mRNAbiao da shui ping 。yi yan zheng zi shi you dao ji shi fou ke yi hui fu yin qiao di TRIM21er yin qi de bing du fu zhi jian shao 。8.5jiang pCAGGS-HA-TRIM21zhen he biao da zhi li zhuai ran A549xi bao 24hhou ,yong zi shi yi zhi ji (wo man qing mei su Wortmannin,100nM)chu li A549xi bao yi yi zhi zi shi shui ping ,zai gan ran RSV。gan ran hou 24htong guo dao zhi ying guang xian wei jing guan cha GFP-RSVbing du ying guang ;yong qPCRjian ce RSV N、RSV F、RSV NS1ji yin mRNAbiao da shui ping 。yi yan zheng zi shi yi zhi ji shi fou ke yi hui fu yin guo biao da TRIM21er yin qi de bing du fu zhi zeng jia 。jie guo :1 RSVgan ran dui A549xi bao TRIM21dan bai biao da de ying xiang 。1.1yong dao zhi ying guang xian wei jing chu bu guan cha GFP-RSVrong ge bing du ji biao qian dan bai GFPzai A549xi bao zhong de biao da 。gan ran RSV 24hhou ,sui zhao gan ran fu shu de zeng jia ,jing xia ying guang ban dian shu liang he liang du you ming xian zeng jia 。qPCRjie guo xian shi ,dang MOI=1shi ,RSVdui TRIM21de you dao zuo yong zui jiang ,ze jiang ji que ding wei zui jia gan ran fu shu 。1.2.RSV(MOI=1)gan ran A549xi bao ,zai gan ran hou 0h、6h、12h、24h、36h、48h,qPCRjie guo xian shi :gan ran hou 12h,TRIM21 mRNAde biao da kai shi you ming xian shang diao ,chi xu sheng gao ,zhi zhi jian ce shi jian dian 48h;Western Blotjie guo biao ming ,gan ran hou 12h,TRIM21dan bai de biao da kai shi you ming xian shang diao ,chi xu sheng gao ,zhi zhi jian ce shi jian dian 48h。ji :RSVgan ran ke you dao TRIM21dan bai gao biao da ,cheng ming xian de shi jian yi lai guan ji 。2cheng gong gou jian pCAGGS-HA-TRIM21zhen he biao da zhi li he pGL4.10-IFNβpromoterying guang su mei bao gao zhi li 2.1cheng gong gou jian pCAGGS-HA-TRIM21zai ti ,jing mei qie he ce xu jian ding zheng que 。2.2cheng gong gou jian pGL4.10-IFNβpromoterying guang su mei bao gao ji yin zai ti ,jing mei qie he ce xu jian ding zheng que 。3li yong duo chong shou duan que ding TRIM21de ya xi bao ding wei 3.1 RSVgan ran A549xi bao 48hhou ,fen li he dan bai he jiang dan bai ,Western blotjian ce jie guo xian shi ,TRIM21dan bai da duo shu biao da zai xi bao jiang zhi zhong 。3.2yong pCAGGS-HA-TRIM21zhen he biao da zhi li zhuai ran pa pian sheng chang de A549xi bao 24hhou ,zai gan ran RSV 24h,mian yi ying guang ran se ,ying guang xian wei jing guan cha jie guo xian shi ,TRIM21dan bai ji hu quan bu fen bu yu xi bao jiang zhi zhong ,cheng pian zhuang san zai fen bu 。4 TRIM21dan bai dui RSVbing du fu zhi de ying xiang 4.1jiang TRIM21de siRNAzhuai ran A549xi bao 24hhou ,zai gan ran RSV,zai gan ran hou 0h,24h,48hshou ji xi bao ,fen bie jin hang qPCRhe Western Blotjian ce 。jie guo xian shi ,yu dui zhao zu xiang bi ,qiao di TRIM21hou ,TRIM21 mRNAji dan bai biao da jun xian zhe xia jiang ,di shi qiao di xiao guo jiao hao 。4.2jiang siTRIM21zhuai ran A549xi bao 24hhou ,zai gan ran RSV,yu gan ran hou 24hhe 48h,shou ji xi bao ,jin hang qPCRhe bing du di du jian ce 。qPCRjie guo xian shi ,yu dui zhao zu xiang bi ,qiao di TRIM21hou ,RSV Nji yin mRNAbiao da you ming xian xia jiang ;bing du di ding (gan ran hou 24h)jie guo xian shi ,yu dui zhao zu xiang bi ,qiao di TRIM21ke ming xian jiang di RSVbing du di du 。4.3jiang pCAGGS-HA-TRIM21zhen he biao da zhi li zhuai ran A549xi bao ,shou ji xi bao ,fen bie jin hang qPCRhe Western blotjian ce 。jie guo xian shi ,yu pCAGGS-HAkong zai zhuai ran zu xiang bi ,pCAGGS-HA-TRIM21zhuai ran zu zhong TRIM21mRNAji dan bai de biao da jun you xian zhe sheng gao ,di shi guo biao da xiao guo jiao hao 。4.4jiang pCAGGS-HA-TRIM21zhen he biao da zhi li zhuai ran A549xi bao 24hhou ,zai gan ran RSV 24h,shou ji xi bao ,jin hang qPCRhe bing du di ding jian ce 。qPCRjie guo xian shi ,yu dui zhao zu xiang bi ,guo biao da TRIM21ke ming xian di gao RSV Nji yin mRNAde biao da ;bing du di ding jie guo xian shi :guo biao da TRIM21ke ming xian di gao bing du di du 。5 TRIM21dan bai dui RSVyou dao de Ⅰxing gan rao su de ying xiang 5.1yong siRNAqiao di A549xi bao TRIM21ji yin de biao da hou ,zai gan ran RSV,gan ran hou 24h,shou ji xi bao ,jin hang qPCR,jie guo xian shi ,qiao jiang TRIM21ke ming xian jiang di Ⅰxing gan rao su ji ji xia you yi ji lie gan rao su ci ji ji yin (ISGs)de mRNAde biao da 。5.2jiang siTRIM21、pGL4.10-IFNβpromoterying guang su mei bao gao zhi li he hai shen ying guang su mei can zhao zhi li pGL4.10-TKgong zhuai ran A549xi bao 24hhou ,zai gan ran RSV,gan ran hou 24h,jin hang shuang ying guang su mei bao gao shi yan 。jie guo xian shi ,qiao jiang TRIM21ke ming xian jiang di gan rao su βqi dong zi huo xing 。5.3jiang pCAGGS-HA-TRIM21zhen he biao da zhi li zhuai ran A549xi bao 24hhou ,zai gan ran RSV 24h,shou ji xi bao ,jin hang qPCRjian ce 。jie guo xian shi ,guo biao da TRIM21ke ming xian shang diao Ⅰxing gan rao su ji xia you yi ji lie ISGsji yin de mRNAde biao da 。5.4jiang pCAGGS-HA-TRIM21zhen he biao da zhi li 、pGL4.10-IFNβpromoterying guang su mei bao gao zhi li he hai shen ying guang su mei can zhao zhi li pGL4.10-TKgong zhuai ran A549xi bao 24hhou ,zai gan ran RSV 24h,jin hang shuang ying guang su mei bao gao shi yan 。jian ce jie guo xian shi ,guo biao da TRIM21ke ming xian zeng jia gan rao su βqi dong zi huo xing 。6 TRIM21yu Ⅰxing gan rao su de xiang hu you dao zuo yong 6.1yong bu tong nong du ti du de ren yuan IFNβchong zu dan bai ci ji A549xi bao 12h,qPCRjie guo xian shi ,yi 5ng/mlji di nong du de ren yuan IFNβchong zu dan bai jiu zu yi you dao TRIM21ji yin de gao biao da ,bu tong ji liang dui TRIM21de you dao zuo yong cha bie bu da 。que ding zui you ji liang hou ,yi gai ji liang de ren yuan IFNβchong zu dan bai ci ji A549xi bao ,fen bie yu 0h,4h,8h,12hhe 16hshou ji xi bao ,jin hang qPCRjian ce 。jie guo xian shi ,ren yuan IFNβchong zu dan bai ci ji A549xi bao hou 4h,ji ke ming xian di gao TRIM21de biao da shui ping ,yu 12hda dao feng zhi 。6.2jiang pCAGGS-HA-TRIM21zhen he biao da zhi li zhuai ran A549xi bao 24hhou ,shou ji xi bao ,jin hang qPCRjian ce 。jie guo xian shi :guo biao da TRIM21ke ming xian di gao IFNβji yin mRNAde biao da shui ping ;jiang pCAGGS-HA-TRIM21zhen he biao da zhi li 、pGL4.10-IFNβpromoterying guang su mei bao gao zhi li he hai shen ying guang su mei can zhao zhi li pGL4.10-TKgong zhuai ran A549xi bao 24hhou ,jin hang shuang ying guang su mei bao gao ji yin jian ce 。jie guo xian shi ,guo biao da TRIM21ke ming xian di gao gan rao su βqi dong zi huo xing 。zeng ge liang ge shi yan jie guo ,di shi :TRIM21can yu diao kong A549xi bao de Ⅰxing gan rao su tong lu 。7 TRIM21ke tong guo gan rao su fei yi lai ji zhi cu jin bing du fu zhi 7.1ying yong siRNAqiao di gan rao su ji yin zi ran que shi de Veroxi bao de TRIM21ji yin biao da zhi hou ,zai gan ran RSV 24h,jin hang Western blothe bing du di ding 。Western blotjie guo xian shi :qiao di TRIM21ming xian jiang di RSVbing du dan bai biao da ;bing du di ding (gan ran hou 24h)jie guo xian shi :qiao di TRIM21ke ming xian jiang di RSVbing du di du 。7.2yong pCAGGS-HA-TRIM21zhen he biao da zhi li zhuai ran Veroxi bao 24hhou ,zai gan ran RSV 24h,shou ji xi bao ,jin hang Western blothe bing du di ding 。Western blotjie guo xian shi :guo biao da TRIM21ke ming xian sheng gao RSVbing du dan bai biao da ;bing du di ding (gan ran hou 24h)jie guo xian shi :guo biao da TRIM21ke ming xian di gao RSVbing du di du 。8 TRIM21ke tong guo zi shi ji zhi cu jin RSVbing du fu zhi 。8.1yi zui jia MOIde RSVgan ran A549xi bao hou ,yu 0h、24h、48hshou ji xi bao ,di qu xi bao zong dan bai ,jin hang Western blot。Western blotjie guo xian shi ,RSVgan ran xi bao hou ,LC3Ⅱdan bai biao da zeng gao ,ji RSVcheng gong you dao le A549xi bao de zi shi ,yi zhi chi xu dao jian ce dian 48h。8.2ying yong siRNAqiao di A549xi bao TRIM21ji yin de biao da hou ,zai gan ran RSV,yu gan ran hou 24h,jin hang Western blot。jie guo xian shi ,mo lun shi fou you RSVgan ran ,qiao di TRIM21hou jun neng jiang di LC3Ⅱdan bai biao da ,di shi qiao di TRIM21biao da ke yi jiang di xi bao zi shi shui ping 。8.3jiang pCAGGS-HA-TRIM21zhen he biao da zhi li zhuai ran A549xi bao 24hhou ,zai gan ran RSV,yu gan ran hou 24h,jin hang Western blot。jie guo xian shi ,mo lun shi fou you RSVgan ran ,guo biao da TRIM21hou jun neng di gao LC3Ⅱdan bai biao da ,di shi guo biao da TRIM21ke yi di gao xi bao zi shi shui ping 。8.4ying yong siRNAqiao di A549xi bao TRIM21ji yin de biao da hou ,yong zi shi you dao ji chu li A549xi bao yi hui fu zi shi shui ping ,zai gan ran RSV。gan ran hou 24hyong dao zhi ying guang xian wei jing chu bu guan cha ,qiao di TRIM21zhi hou ,yu dui zhao zu xiang bi ,GFP-RSVbing du ying guang ban dian shu liang ming xian jian shao ,yu zhi qian jie guo yi zhi 。zai qiao di TRIM21de ji chu shang ,zai jia ru zi shi you dao ji ,GFP-RSVbing du ying guang ban dian shu liang ming xian zeng jia ;qPCRjie guo xian shi ,zi shi you dao ji bu hui ying xiang TRIM21ji yin de biao da ,dan yu chan du qiao di TRIM21zu bi jiao ,zi shi you dao ji ke yi ming xian hui fu RSV N、RSV F、RSV NS1ji yin de biao da shui ping 。di shi :zi shi you dao ji ke yi hui fu yin qiao di TRIM21yin qi de bing du fu zhi jian shao 。8.5jiang pCAGGS-HA-TRIM21zhen he biao da zhi li zhuai ran A549xi bao 24hhou ,yong zi shi yi zhi ji chu li A549xi bao yi yi zhi zi shi shui ping ,zai gan ran RSV。gan ran hou 24hyong dao zhi ying guang xian wei jing chu bu guan cha ,guo biao da TRIM21zhi hou ,yu dui zhao zu xiang bi ,GFP-RSVbing du ying guang ban dian shu liang ming xian zeng jia ,yu zhi qian jie guo yi zhi 。zai guo biao da TRIM21de ji chu shang ,zai jia ru zi shi yi zhi ji ,GFP-RSVbing du ying guang ban dian shu liang ming xian jian shao ;qPCRjie guo xian shi ,zi shi yi zhi ji bu hui ying xiang TRIM21ji yin de biao da ,dan yu chan du guo biao da TRIM21zu bi jiao ,zi shi yi zhi ji ke yi ming xian yi zhi RSV N、RSV F、RSV NS1ji yin de biao da shui ping 。di shi :zi shi yi zhi ji ke yi yi zhi yin guo biao da TRIM21yin qi de bing du fu zhi zeng jia 。jie lun :1.RSVgan ran ke yi shang diao A549xi bao zhong TRIM21 mRNAji dan bai de biao da 。2.TRIM21dan bai zhu yao ding wei yu xi bao jiang zhong 。3.TRIM21dan bai ke yi cu jin RSVfu zhi 。4.TRIM21yu Ⅰxing gan rao su ke yi xiang hu you dao ,bing hen ke neng cun zai yi ge zheng fan kui huan lu 。5.TRIM21dan bai ke tong guo gan rao su fei yi lai ji zhi cu jin RSVfu zhi 。6.TRIM21dan bai ke tong guo zi shi ji zhi cu jin RSVfu zhi 。
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