DJ-1介导缺氧心肌HL-1细胞线粒体动力学变化

Effect of Down-regulation of DJ-1 on Mitochondrial Dynamics of Hypoxia HL-1 Mouse Cardiomyocytes

目的探讨DJ-1参与缺氧心肌细胞线粒体动力学改变的机制。方法采用慢病毒载体shRNA DJ-1感染心肌HL-1细胞株,通过流式细胞术、激光共聚焦显微镜、线粒体-细胞质亚结构分离技术和Westernblot观察缺氧后线粒体融合-分裂蛋白DRP1、MFN1和MFN2蛋白表达、线粒体膜电位及线粒体形态变化。结果正常和缺氧培养时,稳定表达shRNA DJ-1的HL-1细胞DJ-1蛋白表达均显著下调,而在缺氧培养时shRNA DJ-1细胞的线粒体标志蛋白TOM20蛋白表达显著下调。流式细胞仪结果表明,空慢病毒对照缺氧组相比正常空慢病毒对照组线粒体膜电位下调,而shRNA DJ-1加剧这种线粒体膜电位下调。分离缺氧培养心肌HL-1细胞线粒体,线粒体分裂蛋白DRP1和融合蛋白MFN2表达均增加。结论缺氧条件下DJ-1是线粒体动力学平衡稳定的因素。

Objective To explore the potential mechanism of protective effect of DJ-1 on mitochondrial dynamics in hypoxia heart.Methods Down-regulation of DJ-1 transfected HL-1 mouse cardiomyocyte strain with lentiviral vector shRNA.The mitochondrial protein expression was measured with Western blot analysis,mitochondrial membrane potential（△ψm）and mitochondrial dynamics（its fusion-fission protein DRPI,NFWI and MFN2 protein expression,as well as its morphological changes） were assessed with flow cytometry,confocal microscope and mitochondrion-cytoplasm separation techniques.Results The efficiency of lentiviral vector shDJ-1 silencing DJ-1 protein expression was significantly decreased in normal culture and hypoxia culture group.Mitochondria marker TOM20 protein expression of lentiviral vector shDJ-1 was significantly decreased under hypoxia culture.It was showed with flow cytometry that mitochondrial membrane potential of lentiviral vector shDJ-1 was decreased under hypoxia culture and intensified by shRNA DJ-1.The lentiviral vector shDJ-1 mitochondrial proteins expression of separated HL-1 mitochondria fission protein DRP1 and fusion protein MFN2 were increased under hypoxia culure.Conclusion DJ-1 stablizes mitochondrial dynamics under hypoxia.