c-Abl基因在Ang Ⅱ诱导血管平滑肌细胞氧化应激中的作用  

Role of c-Abl Gene in Ang Ⅱ-induced Oxidative Stress of Vascular Smooth Muscle Cells

作　　者：孙图成[1] 周先吾 李华东[2] 陈澍[2] 许菲[3] 蒋雄刚[2] 

机构地区：[1]广东省心血管病研究所广东省人民医院心血管外科广东省医学科学院,广州510080 [2]华中科技大学同济医学院附属协和医院心血管外科,武汉430022 [3]武汉大学附属中南医院消化内科,武汉430071

出　　处：《华中科技大学学报（医学版）》2017年第4期380-385,共6页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家自然科学基金资助项目(No.81370416)

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、(2)AngⅡ组、(3)STI571组、(4)lenti-control组、(5)lenti-cAbl-shRNA组、(6)lenti-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(1×10-7 mol/L)干预30min,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(1×10-7 mol/L)干预36h,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pcAbl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pcAbl蛋白水平无明显差异。AngⅡ干预36h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 angiotensin Ⅱ （Ang Ⅱ ）-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, Ang Ⅱ group, STI571 group, lenti-control group, lenti-cAbl-shRNA group, lenti-cAbl group （groups 1-6）. Groups 1-3 were normal krSMCs,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 （〈 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 Ang Ⅱ （1 X 10 7 mol/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 Ang Ⅱ group. ROS,DHE and DHR levels increased after 36 h of Ang Ⅱ stimulation,but the oxidative stress level of lenti-cAbl group was the highest among all the groups. Compared with the Ang Ⅱ 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 oxidat

关 键 词：慢病毒载体 c-Abl基因 血管平滑肌细胞 氧化应激 血管紧张素Ⅱ 

分 类 号：R543.1[医药卫生—心血管疾病]