肠道病毒71型对人神经胶质瘤U251细胞线粒体动力学的影响

张琬棂; 刘正芸; 王盛羽; 罗果; 易海波; 宋鸿; 王欢

doi:10.12182/20200360201

肠道病毒71型对人神经胶质瘤U251细胞线粒体动力学的影响

Effects of Enterovirus 71 on Mitochondrial Dynamics in Human Glioma U251 Cells

摘要

摘要:
目的探讨肠道病毒71型（EV71）对人神经胶质瘤U251细胞线粒体动力学的影响。
方法采用非洲绿猴肾细胞Vero对EV71进行增殖，用Reed-Muench公式计算病毒液的50%细胞感染剂量（50% tissue culture infective dose，TCID₅₀)；将EV71接种于U251细胞，光学显微镜下观察细胞形态，激光共聚焦显微镜下观察线粒体形态，透射电镜观察线粒体超微结构的改变；Western blot检测线粒体分裂蛋白Drp1、p-Drp1(S637)和融合蛋白Opa1的表达水平；ATP试剂盒检测线粒体ATP水平；MitoSOX染色检测线粒体超氧化物水平。
结果 EV71的TCID₅₀为10^－5.4/ 0.1 mL； EV71感染U251细胞24 h、48 h后细胞明显出现皱缩、变圆或死亡。激光共聚焦显微镜和透射电镜结果显示，EV71感染后细胞线粒体明显延长，线粒体嵴模糊不清；Western bolt结果显示，EV71感染U251细胞24 h和48 h后，Drp1、Opa1表达降低，而p-Drp1（S637）在48 h表达升高，且差异有统计学意义（P<0.05）。EV71感染48 h后，与未感染EV71的U251细胞相比，线粒体ATP生成减少，线粒体超氧化物水平升高（P<0.05）。
结论 EV71感染U251细胞后，能引起线粒体形态及动力学改变，进而导致线粒体功能的变化，这可能是其引起神经系统功能紊乱的机制之一。

Abstract:
Objective To investigate the effects of enterovirus 71 (EV71) on mitochondrial dynamics in human Glioma U251 cells.
Methods The EV71 was replicated in Vero cells and the 50% tissue culture infective dose (TCID₅₀) was calculated based on the Reed-Muench formula. After the U251 cells were infected with EV71, the cellular morphology was assessed through the light microscope. The mitochondrial morphology was detected by MitoTracker Deep Red staining under laser confocal microscopy and the mitochondrial ultrastructure was visualized by transmission electron microscopy. The expressions of mitochondrial fission proteins Drp1, p-Drp1 and fusion protein Opa1 were examined by Western blot. The level of ATP was measured by a commercial ATP assay kit. The generation of mitochondrial superoxide was detected by MitoSOX staining.
Results The TCID₅₀ of EV71 was 10^－5.4/0.1 mL. Twenty-four or 48 h after EV71 infection, the U251 cells appeared shrunken, round and dead. The laser confocal microscopy and transmission electron microscopy images showed that the EV71 infection induced mitochondrial elongation and cristae damage. Moreover, Western blot analysis demonstrated that the protein expressions of Drp1 and Opa1 were downregulated at both 24 and 48 h after EV71 infection in U251 cells, companied with a significant increase in Drp1 phosphorylation at 48 h after infection (P<0.05). In addition, a decreased ATP level and elevated mitochondrial superoxide generation were observed in the EV71 infected group, as compared to the control group.
Conclusion Our study demonstrated that infection with EV71 led to changes of mitochondrial morphology and dynamics in U251 cells, which may impair mitochondrial function and contribute to nervous system dysfunction.

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