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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 study the mechanism of killing primary germ cell of gerbil with enterovirus 71 (EV71) and provide basis for the study on the mechanism of EV71 infection. Methods Primary cultured gerbil muscle cells were isolated by trypsin / collagenase digestion, and the cells were infected with EV71. The viability of infected cells was detected by CCK-8 assay. The nuclear changes of infected cells were detected by Hoechst 33258 staining. Annexin V / PI double staining The changes of cell membrane in infected cells were detected by Western blotting. The expressions of PARP-1, Caspase-3, Bcl-2 family proteins and NF-κB pathway-related proteins in infected cells were detected by Western blotting. Results The germ cell model of gerbil in vitro was constructed. After infected with EV71, the morphological changes of the muscle cells such as shrinkage, rounding and floating were observed. The greater the multiplicity of infection (MOI), the more obvious the degree of lesion, the more the cell survival rate (Rs = -0.964, P = 0.005). Concentration of intracellular chromatin, phosphatidylserine eversion, accompanied with apoptotic bodies, and apoptosis were positively correlated with MOI (rs = 1.000, P <0.001) The apoptotic marker protein PARP-1 and the apoptotic executive protein Caspase-3 were cleaved and activated. The expressions of the anti-apoptotic proteins Bcl-xl and Bcl-2 decreased with the increase of MOI. The expression of p65 protein decreased. The level of NF-κB pathway is activated. Conclusion EV71 virus can induce primary germ cell apoptosis in gerbil.