【摘要】 肠道病毒71型(enterovirus type 71,EV71)感染常可引起婴幼儿手足口病(hand,foot and mouth disease,HFMD),还可引起中枢神经系统并发症等重症,甚至死亡。研究认为,EV71诱发重症的原因主要与病毒感染诱导细胞程序性死亡(programmed cell death,PCD)及诱导细胞产生大量炎症因子有关。病毒感染可通过激活不同的信号通路触发细胞程序性死亡,主要包括含半胱氨酸的天冬氨酸蛋白水解酶(cysteinyl aspartate specific proteinase,caspase)依赖的细胞凋亡、细胞焦亡,以及非caspase依赖的细胞坏死性凋亡。本研究旨在探讨EV71感染诱导细胞程序性死亡的形态学和分子生物学特征,利用显微镜和免疫荧光技术检测EV71感染后细胞形态变化,JC-1染色检测感染后细胞线粒体膜电位变化,流式细胞术及Annexin VFITC/PI双染法、乳酸脱氢酶释放量法检测感染细胞的细胞膜损伤程度,结合蛋白免疫印迹法检测病毒感染后细胞中多聚ADP核糖聚合酶[poly(ADP-ribose)polymerase,PARP]、caspase-9、caspase-3等凋亡因子,以及细胞焦亡关键效应蛋白Gasdermin D、坏死性凋亡效应蛋白MLKL的磷酸化情况。结果显示,EV71感染后细胞主要呈现凋亡特征,并伴随少量细胞坏死。与细胞凋亡相关的PARP被剪切,caspase-9和caspase-3等相关因子被激活。经泛caspase抑制剂处理后,细胞程序性死亡被抑制,但仍有部分细胞坏死。结果提示,EV71感染以细胞凋亡为主,也可能存在非caspase依赖的细胞程序性死亡。更多还原

【Abstract】 Enterovirus type 71(EV71)infection usually causes hand,foot and mouth disease(HFMD)in infants and young children.Severe EV71 infections can result in central nervous system complications,and even death.Previous studies suggest that EV71-induced severe complications are associated with programmed cell death and production of pro-inflammatory factors.To determine the molecular morphology and characteristics of EV71-induced cell death,EV71-infected cells were subjected to morphological analysis,JC-1 staining for potential of mitochondrial membrane,fluorescence-activated cell sorting,Annexin VFITC/PI double staining,and lactate dehydrogenase(LDH)release assay.Western blotting was used to identify the activation of programmed cell death factors,including poly(ADP-ribose)polymerase(PARP),caspase-9,caspase-3,Gasdermin D,and MLKL.The results showed that EV71-infected cells mainly induced apoptotic cell death with a small portion of cellular necrosis.The cleavage of PARP,caspase-9,caspase-3 was observed.Although cell death was mostly inhibited by a caspase inhibitor,cell death still could be observed during the early stage of virus infection.The results suggest that EV71 infection mainly induces apoptotic cell death in combination with a caspase-independent cell programmed death.更多还原