肠道病毒71型诱导沙鼠肌肉细胞凋亡及其机理研究

The mechanism of apoptosis in muscle cells from gerbils induced by Enterovirus 71

目的研究肠道病毒71型(EV71)杀伤沙鼠原代肌肉细胞的作用机理,为EV71的感染机制研究提供依据。方法采用胰酶/胶原酶消化法分离培养沙鼠原代肌肉细胞,滴加EV71感染细胞,采用CCK-8法检测感染细胞存活率,Hoechst 33258染色法检测感染细胞核变化,Annexin V/PI双染法检测感染细胞的细胞膜变化,蛋白免疫印迹试验检测感染细胞的PARP-1、Caspase-3、Bcl-2家族蛋白等凋亡因子和NF-κB通路相关蛋白表达量的变化。结果构建体外沙鼠原代肌肉细胞模型,使其感染EV71后,肌肉细胞出现皱缩、变圆、漂浮等形态学变化,且感染复数(MOI)越大,病变程度越明显,细胞存活率越低(rs=-0.964,P=0.005);细胞内染色质发生浓缩,细胞膜磷脂酰丝氨酸外翻,伴随凋亡小体出现,细胞凋亡与MOI呈正相关(rs=1.000,P<0.001);凋亡标志蛋白PARP-1与凋亡执行蛋白Caspase-3被剪切激活;抑凋亡蛋白Bcl-xl与Bcl-2的表达量随着MOI的增大而减少;p65蛋白表达水平降低,磷酸化水平升高,NF-κB通路被激活。结论 EV71病毒可以诱导沙鼠原代肌肉细胞发生凋亡。

Objective To explore the mechanism of death induced by EV71 virus in primary muscle cells of gerbils,and to lay foundations on the research of infection mechanism of EV71 virus. Methods The primary muscle cells were isolated and cultured by digestion of tyrisin / collagenase. Survival rates of primary muscle cells infected by EV71 virus were detected by CCK- 8 assays. Changes of the nuclei in infected cells were observed by Hoechst 33258 staining. The redistribution of phosphatidylserine from the inner to the outer leaflet of the plasma membrane was measured by Annexin V / PI double staining. Expression changes of proteins PARP- 1,Caspase- 3,Bcl- xl,Bcl- 2 and p65 were detected by Western Blot.Results We established a primary muscle cells model in vitro successfully. Primary muscle cells form gerbils would be shrank,round and floated in the culture medium after infected by EV71 virus. Several events in apoptosis such as nuclear condensation,phosphatidylserine translocated to the outside of the plasma membrane,cleavage of apoptosis- related proteins PARP- 1 and Caspase- 3 were detected. Further studies showed that EV71 infection could not only activate the NF- κB pathway,but also suppress the apoptosis- suppressing proteins Bcl- xl and Bcl- 2,leading apoptosis in primary muscle cells. Conclusion EV71 could induce apoptosis in primary muscle cells from gerbils.