锌指抗病毒蛋白对肠道病毒A组71型感染引起的细胞焦亡和自噬的实验研究  

Experimental study on the effect of zinc finger antiviral protein on pyroptosis and autophagy induced by enterovirus 71 infection

作　　者：邵华 朱英斌 房庆军 张俊国 SHAO Hua;ZHU Yingbin;FANG Qingjun;ZHANG Junguo(Department of Infectious Diseases,Jinan People's Hospital,Jinan,Shandong 271199,China;不详)

机构地区：[1]济南市人民医院医院感染管理科,山东济南271199 [2]济南市人民医院健康管理科,山东济南271199 [3]济南市人民医院检验科,山东济南271199 [4]山东第一医科大学附属省立医院检验科,山东济南250000

出　　处：《中国病毒病杂志》2024年第6期563-570,共8页Chinese Journal of Viral Diseases

基　　金：济南市卫生健康委员会科技发展计划项目(2024505016)。

摘　　要：目的探讨锌指抗病毒蛋白对肠道病毒A组71型(EV-A71)感染引起的细胞焦亡和自噬的影响。方法将人正常胃上皮细胞(GES-1)细胞随机分为对照组(不感染EV-A71病毒)和感染组(感染EV-A71病毒),采用细胞计数试剂盒-8(CCK-8)检测细胞活力,RT-PCR检测细胞中VP1 mRNA表达,采用二噁英染色(Dil染色)和4',6-二氨基-2-苯基吡啶核染色(DAPI核染色)检测细胞数量和形态。将GES-1细胞随机分为对照组(不感染EV-A71病毒)、EV-A71组(用MOI=5感染的EV-A71感染GES-1细胞)和pc DNA3.1-ZAP组(在EV-A71组的基础上转染pc DNA3.1-ZAP质粒)。测定细胞培养半数感染量(CCID50),ELISA检测细胞培养液中IL-18和IL-1β水平,CCK-8检测细胞活力,蛋白质印迹法检测细胞中自噬相关蛋白LC3、p62及焦亡相关蛋白NLRP3、caspase-1、GSDMD蛋白表达。结果CCK-8检测结果显示,随着MOI和感染时间的增加,EV-A71对GES-1细胞增殖活力的抑制作用越显著。和对照组比较,EV-A71感染GES-1细胞后细胞中VP1 mRNA表达[(2.14±0.21)比(1.00±0.10),t=12.01,P<0.01]明显增加,细胞数量明显减少,细胞核固缩且大小不均。和EV-A71组相比,pc DNA3.1-ZAP组上清液中EV-A71病毒滴度明显降低[(5.21±0.25)比(7.32±0.41),t=10.76,P<0.01]。和对照组比较,EV-A71组细胞培养上清液中IL-18[(64.32±7.18)pg/ml比(20.16±2.32)pg/ml,t=14.34,P<0.01]、IL-1β[(79.38±8.05)pg/ml比(24.11±3.16)pg/ml,t=15.65,P<0.01]水平、细胞中LC3Ⅱ/LC3Ⅰ比值[(1.42±0.14)比(1.00±0.10),t=5.980,P=0.01]、NLRP3[(1.12±0.15)比(0.25±0.03),t=13.93,P<0.01]、caspase-1[(0.74±0.08)比(0.32±0.04),t=11.50,P<0.01]、GSDMD[(0.49±0.06)比(0.26±0.04),t=7.813,P<0.01]蛋白表达均明显升高,细胞活力[(62.31±3.86)%比(100.00±10.00)%,t=8.613,P<0.01]、p62蛋白表达[(0.21±0.04)比(0.98±0.10),t=17.51,P<0.01]明显降低;和EV-A71组相比,pc DNA3.1-ZAP组细胞培养上清液中IL-18[(32.09±4.05)pg/ml比(64.32±7.18)pg/ml,t=9.577,P<0.01]、IL-1β[(39.26±4.12)pg/ml比(79.38±8.05)pg/Objective To investigate the impact of zinc finger antiviral protein(ZAP)on pyroptosis and autophagy induced by enterovirus A71(EV-A71)infection.Methods Human gastric epithelial cells(GES-1)were randomly divided into a control group(uninfected)and an infection group(infected with EV-A71 virus).Cell viability was detected using the cell counting kit-8(CCK-8),and VP1 mRNA expression was detected using RT-PCR.Cell morphology and counts were detected using Dil staining and DAPI nuclear staining.GES-1 cells were randomly divided into control group(uninfected),EV-A71 group(infected with EV-A71 with MOI=5),and pcDNA3.1-ZAP group(transfected with pcDNA3.1-ZAP plasmid in the EV-A71 group).The cell culture median infective dose of 50%(CCID50)was measured,and levels of IL-18 and IL-1βin the culture supernatant were detected by ELISA.Protein expression of autophagy-related proteins(LC3,p62)and pyroptosis-related proteins(NLRP3,caspase-1,GSDMD)was analyzed using Western blotting.Results CCK-8 results showed that the inhibitory effect of EV-A71 on the proliferation viability of GES-1 cells became more significant with increasing MOI and duration of infection.Compared to the control group,the expression of VP1 mRNA in GES-1 cells infected with EV-A71 increased significantly[(2.14±0.21)vs(1.00±0.10),t=12.01,P<0.01],while cell count decreased significantly,and nuclei exhibited shrinkage and irregular sizes.Compared with the EV-A71 group,the titer of EVA71 virus in the supernatant of the pcDNA3.1-ZAP group was significantly reduced[(5.21±0.25)vs(7.32±0.41),t=10.76,P<0.01].Additionally,the levels of IL-18[(64.32±7.18)pg/ml vs(20.16±2.32)pg/ml,t=14.34,P<0.01]and IL-1β[(79.38±8.05)pg/ml vs(24.11±3.16)pg/ml,t=15.65,P<0.01],as well as the LC3Ⅱ/LC3Ⅰratio[(1.42±0.14)vs(1.00±0.10),t=5.980,P=0.01],NLRP3[(1.12±0.15)vs(0.25±0.03),t=13.93,P<0.01],caspase-1[(0.74±0.08)vs(0.32±0.04),t=11.50,P<0.01],and GSDMD[(0.49±0.06)vs(0.26±0.04),t=7.813,P<0.01]were significantly elevated in the EV-A71 group compared to the control grou

关 键 词：锌指抗病毒蛋白 肠道病毒A组71型 细胞焦亡 自噬 

分 类 号：R512.5[医药卫生—内科学]