猪传染性胃肠炎病毒诱导PK-15细胞自噬及其机理  

Mechanism of autophagy in PK-15 cells induced by porcine transmissible gastroenteritis virus

作　　者：刘莹[1] 袁广富 张增贤 张毅 王晶[1] 郭禹 赵云环 范京惠[1] 左玉柱[1] LIU Ying;YUAN Guangfu;ZHANG Zengxian;ZHANG Yi;WANG Jing;GUO Yu;ZHAO Yunhuan;FAN Jinghui;ZUO Yuzhu(College of Veterinary Medicine,Hebei Agricultural University,Boading,Hebei 0710ol,China;Agriculture and Rural Affairs Bureau of Ningjin County,Ningjin,Hebei 055550,China;Disease Control and Prevention in Xingtai,Xingtai,Hebei 054000,China)

机构地区：[1]河北农业大学动物医学院,河北保定071001 [2]宁晋县农业农村局,河北宁晋055550 [3]邢台市动物疫病预防控制中心,河北邢台054000

出　　处：《中国兽医学报》2022年第7期1350-1355,1456,共7页Chinese Journal of Veterinary Science

基　　金：河北省重点研发计划资助项目(19226622D);河北省农业产业技术体系生猪创新团队资助项目(HBCT2018110207)。

摘　　要：首先利用间接免疫荧光方法、Western blot及Image J灰度值分析,研究猪传染性胃肠炎病毒(transmissible gastroenteritis virus,TGEV)感染细胞后及通过紫外线灭活后的病毒对自噬相关蛋白LC3的蛋白表达及转化水平的影响;其次利用自噬促进剂雷帕霉素及自噬抑制剂3-MA对PK-15细胞进行处理,通过qRT-PCR及病毒滴度测定研究细胞自噬在其病毒增殖过程中所产生的影响;最后通过qRT-PCR对自噬关键基因在病毒感染细胞时的动态变化及影响进行分析。结果显示,随着病毒感染时间的增加,细胞内的LC3-Ⅰ逐渐向LC3-Ⅱ转化,其LC3-Ⅱ/β-actin的比值明显增加;结合雷帕霉素作用后,细胞自噬极显著上调了TGEV mRNA转录水平及病毒滴度(P<0.01);与自噬抑制剂3-MA结合后,随病毒感染时间的不同,极显著下调了其病毒的mRNA转录量及病毒滴度(P<0.01)。而TGEV在感染细胞后极显著诱导了自噬关键基因(ATG-5及Beclin-1)mRNA转录水平的提升(P<0.01),并结合病毒增殖情况印证了细胞在受到TGEV感染后,其病毒增殖与自噬程度呈正向作用,说明病毒感染可诱导细胞自噬,促进细胞自噬会增加分离株的病毒滴度。结果表明,TGEV感染PK-15细胞后可诱导细胞自噬,并确定了自噬在TGEV感染中的重要作用。The study firstly used indirect immunofluorescence method,Western blot and ImageJ gray value analysis to study the effect of transmissible gastroenteritis virus(TGEV)and the virus inactivated by ultraviolet light on the protein expression and transformation level of autophagy-related protein LC3 in infected cells.Secondly,PK-15 cells were treated with autophagy enhancer rapamycin and autophagy inhibitor 3-MA to study the effect of autophagy on the proliferation of TGEV by quantitative fluorescence detection method and virus titer determination.Finally,the quantitative fluorescence detection method was used to analyze the dynamic changes and effects of autophagy associated genes when the virus infected cells.The study found that with the increase of virus infection time,the LC3-Ⅰgradually transformed into LC3-Ⅱ,and the ratio of LC3-Ⅱ/β-actin increased significantly.After combined with rapamycin,autophagy significantly up-regulated TGEV mRNA transcription and virus titer(P<0.01).When combined with autophagy inhibitor 3-MA,the mRNA transcription amount and virus titer were significantly down-regulated with the time of virus infection(P<0.01).After the cells were infected with the TGEV HB-FP strain,the mRNA transcripts of the key autophagy genes Beclin-1and ATG-5were significantly increased in the virus-infected cells,showing a very significant difference(P<0.01).After the cells were infected with the TGEV HB-FP strain,the mRNA transcripts of the key autophagy genes Beclin-1and ATG-5were significantly increased in the virus-infected cells,and combined with the results of virus proliferation,it was confirmed that the positive effect of virus proliferation on the degree of autophagy in cells infected with TGEV,indicating that virus infection can induce cellular autophagy;promoting cellular autophagy increases the virus titer of the isolates.This study proved that TGEV infection of PK-15cells could induce autophagy,and confirmed the important role of autophagy in TGEV infection.

关 键 词：细胞自噬 TGEV 病毒增殖 自噬相关基因 雷帕霉素 3-MA 

分 类 号：S852.3[农业科学—基础兽医学] S852.65[农业科学—兽医学]