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目的观察高糖缺氧状态人脐静脉内皮细胞(HUVECs)自噬水平的变化,探讨自噬在这一过程中所起到的作用。方法通过体外细胞培养的方法培养HUVECs并分为6组,即正常对照组、高糖组、正常细胞缺氧组、高糖缺氧组、高糖缺氧+雷帕霉素组、高糖缺氧+3-甲基腺嘌呤(3-MA)组。各组经过相应处理后,通过流式细胞仪、Caspase-3活性试剂盒检测凋亡率、Western blot检测自噬相关蛋白(LC-3、Beclin-1、Atg-5和STSQM1)的表达情况。结果与正常缺氧组相比,高糖缺氧组的自噬相关蛋白LC-3B、Beclin-1和Atg-5表达降低,STSQM1表达升高(均P<0.05),使用雷帕霉素可以上调高糖缺氧组自噬水平并且降低细胞凋亡率,加入自噬抑制剂3-MA使高糖缺氧组自噬下调,细胞凋亡率上升(均P<0.05)。结论高糖缺氧状态下HUVECs自噬功能受损,上调自噬可以降低凋亡率,发挥保护细胞的作用。
Objective To observe the changes of autophagy in human umbilical vein endothelial cells (HUVECs) under hypoglycemic conditions and explore the role of autophagy in this process. Methods HUVECs were cultured by in vitro cell culture and divided into 6 groups: normal control group, high glucose group, normal cell hypoxia group, high glucose hypoxia group, high glucose hypoxia + rapamycin group, Oxygen + 3-methyladenine (3-MA) group. After corresponding treatment, the apoptotic rate was detected by flow cytometry and Caspase-3 activity kit, and the expressions of autophagy-related proteins (LC-3, Beclin-1, Atg-5 and STSQM1) were detected by Western blot. Results Compared with normal hypoxia group, the expressions of autophagy-related protein LC-3B, Beclin-1 and Atg-5 were decreased and STSQM1 expression was increased (all P <0.05) (P <0.05). Autophagy was upregulated and the rate of apoptosis was decreased. Autophagy inhibitor 3-MA was used to down-regulate autophagy in high glucose and hypoxia groups. Conclusion HUVECs are impaired in autophagy under high glucose and hypoxia conditions. Upregulation of autophagy can reduce the apoptosis rate and play a role in protecting cells.