缺氧环境下自噬对内皮祖细胞迁移及凋亡的影响

Effects of autophagy on cell migration and apoptosis of endothelial progenitor cells in hypoxic environment

目的探讨缺氧环境下自噬对大鼠内皮祖细胞(EPCs)迁移、凋亡的影响。方法采用密度梯度离心法分离获取EPCs并用荧光双染法鉴定大鼠骨髓源性EPCs。为检测缺氧对EPCs迁移、凋亡及自噬水平的影响,将细胞分为4组:常氧培养组(即缺氧0h组)、缺氧1h组、缺氧3h组和缺氧6h组,用Transwell小室和流式细胞术分别检测各组细胞迁移和凋亡情况,Western blotting检测EPCs自噬相关蛋白LC3-Ⅱ/Ⅰ和p62的改变。为检测沉默自噬后缺氧对EPCs的影响,将细胞分为4组:缺氧0h组、缺氧3h组、缺氧3h+沉默Atg7(Sh Atg7)组和Sh Atg7组,再次观察EPCs迁移及凋亡的变化。结果与缺氧0h组比较发现,缺氧3h及6h后EPCs迁移能力明显降低(P<0.05),细胞凋亡率明显升高(P<0.05);且缺氧3h及6h后EPCs内自噬相关蛋白LC3-Ⅱ/Ⅰ的表达明显升高(P<0.05),p62表达明显降低(P<0.05)。Sh Atg7抑制自噬后发现,与缺氧3h组比较,缺氧3h+Sh Atg7组的EPCs迁移能力进一步降低(P<0.05),细胞凋亡率进一步升高(P<0.05)。结论缺氧环境下自噬可促进EPCs迁移并减少凋亡,可对EPCs存活发挥保护作用。

Objective To investigate the effects of autophagy on cell migration and apoptosis of endothelial progenitor cells（EPCs） in hypoxic environment. Methods Rats＇ bone marrow-derived EPCs were isolated by density-gradient centrifugation, identified with double-fluorescent staining method, and then divided into 4 groups： normoxia（hypoxia 0 h）, hypoxia 1 h, hypoxia 3 h and hypoxia 6 h groups for detecting the effects of hypoxia on EPCs migration and apoptosis. Transwell chambers and flow cytometry were used to detect cell migration and apoptosis respectively, and Western blotting was performed to detect the changes of autophagy-related proteins, including microtubule associated protein 1 light chain 3（LC3-Ⅱ/Ⅰ） and ubiquitin of p62. To investigate the effects of hypoxic-induced autophagy on EPCs migration and apoptosis, cells were divided into 4 groups： hypoxia 0 h, hypoxia 3 h, hypoxia 3 h＋Sh Atg7 and Sh Atg7 groups, and then to detect again the EPCs migration and apoptosis. ResultsCompared to hypoxia 0 h group, the EPCs migration markedly reduced and apoptosis increased in hypoxic 3 h and 6 h groups with statistically significant difference（P〈0.05）. Western blotting showed the expressions of LC3-Ⅱ/Ⅰ significantly increased and of p62 decreased in EPCs by hypoxic treatment for 3 and 6 hours（P〈0.05）, implying the increases of autophagy level. After inhibiting autophagy by silencing Atg7, hypoxia 3 h＋Sh Atg7 group further reduced EPCs migration（P〈0.05） and aggravated apoptosis（P〈0.05） compared with hypoxia 3 h group. Conclusion Autophagy may play a cytoprotective role through promoting EPCs migration and decreasing EPCs apoptosis in hypoxic environment.