c-Abl基因在AngⅡ诱导血管平滑肌细胞氧化应激中的作用

摘要

目的 探讨c-Abl基因在血管紧张素Ⅱ(AngⅡ)诱导血管平滑肌细胞(VSMCs)氧化应激中的作用.方法 构建c-Abl基因过表达和沉默的慢病毒载体,并利用慢病毒转染的方法构建c-Abl过表达和沉默的VSMCs.实验分6组:①对照组、②AngⅡ组、③STI571组、④lenti-control组、⑤lenti-cAbl-shRNA组、⑥lenti-cAbl组.①~③组为正常VSMCs,③组用STI571预处理,④~⑥组为分别利用lenti-control、lenti-cAbl-shRNA、lenti-cAbl转染VSMCs,对照组用生理盐水干预,其余实验组用AngⅡ(1×10-7mol/L)干预30 min,并用Western blot测定c-Abl和p-cAbl蛋白表达量;活性氧测定:对照组用生理盐水干预,其余实验组用AngⅡ(1×10-7mol/L)干预36 h,流式细胞仪分别检测其活性氧分子(ROS)、二氢罗丹明(DHR)、二氢乙啶(DHE)水平.结果 AngⅡ刺激后,与对照组比较,lenti-cAbl组的c-Abl和p-cAbl蛋白表达最强,STI571组和lenti-cAbl-shRNA组p-cAbl蛋白表达最弱,而lenti-control组与AngⅡ组c-Abl和p-cAbl蛋白水平无明显差异.AngⅡ干预36 h后,VSMCs氧化应激产物(ROS、DHE、DHR)均增加,但lenti-cAbl组氧化应激水平最高,lenti-cAbl-shRNA组、STI571组的氧化应激水平较AngⅡ组有所降低.结论 利用慢病毒载体构建过表达c-Abl基因的VSMCs细胞系获得成功;c-Abl基因表达的增加与AngⅡ刺激下VSMCs的氧化应激程度呈正相关.%Objective To investigate the role of c-Abl gene in angiotensinⅡ(AngⅡ)-induced oxidative stress in vascular smooth muscle cells(VSMCs).Methods We constructed the c-Abl gene overexpression and silence lentiviral vectors and used lentivirus transfection method to construct c-Abl overexpression and silence VSMCs.The test group VSMCs were divided into six groups:control group,AngⅡ group,STI571 group,lenti-control group,lenti-cAbl-shRNA group,lenti-cAbl group (groups 1-6).Groups 1-3 were normal VSMCs,Group 3 was pretreated with STI571,and groups 4-6 were VSMCs transfected by lenti-control,lenti-cAbl-shRNA and lenti-cAbl,respectively.the control group was treated with normal saline for 30 min,while the other experimental groups were treated with AngⅡ(1×10-7 mol/L)for 30 min,then we used Western blotting to detect the protein expression of c-Abl and p-cAbl.For determination of reactive oxygen species(ROS),the control group was treated with normal saline,while the other experimental groups were treated with AngⅡ(1×10-7mol/L)for 36 h,then flow cytometry was used to detect the oxidative stress levels of reactive oxygen species(ROS),dihydroethidium(DHE)and dihydrorhodamine(DHR),respectively.Results Compared with the control group,the expression of p-cAbl protein was the highest in the lenti-cAbl group and the weakest in STI571 and lenti-cAbl-shRNA groups.There were no significant differences in the levels of c-Abl and p-cAbl protein between lenti-control group and AngⅡ group.ROS,DHE and DHR levels increased after 36 h of AngⅡ stimulation,but the oxidative stress level of lenti-cAbl group was the highest among all the groups.Compared with the AngⅡ group,the oxidative stress levels of lenti-cAbl-shRNA group and STI571 group were reduced.Conclusion The VSMCs with the overexpressing c-Abl gene was successfully constructed by lentiviral vector technique.The increase of c-Abl gene expression was positively correlated with the oxidative stress of VSMCs stimulated by AngⅡ.