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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 investigate the mechanism of DJ-1 involved in the mitochondrial dynamics of hypoxic cardiomyocytes. Methods The HL-1 cells were infected with lentiviral vector shRNA DJ-1. The mitochondrial fusion-cleavage proteins DRP1 and MFN1 were detected by flow cytometry, confocal laser scanning microscopy, mitochondria-cytoplasm subtype separation and Western blotting. MFN2 protein expression, mitochondrial membrane potential and mitochondrial morphological changes. Results DJ-1 protein expression was significantly down-regulated in HL-1 cells stably expressing shRNA DJ-1 at normal and hypoxic conditions, whereas the expression of mitochondrial protein TOM20 was down-regulated in shRNA DJ-1 cells under hypoxia. Flow cytometry results showed that compared with normal lentivirus control group, empty lentiviral control hypoxic group mitochondrial membrane potential down regulation, and shRNA DJ-1 aggravated the mitochondrial membrane potential down regulation. Mitochondria were isolated from hypoxia-cultured myocardial HL-1 cells and the expression of mitochondrial mitochondrial DRP1 and fusion protein MFN2 were increased. Conclusion DJ-1 is an important factor in the stable and stable mitochondrial dynamics under hypoxia.