内皮素1过表达经Akt／PKB信号通路抑制大鼠肺细小动脉平滑肌细胞凋亡的体外研究  

Endothelin-1 overexpression inhibits rat pulmonary arterial microvascular smooth muscle cell apoptosis via Akt/PKB pathway

作　　者：黄海琼[1] 王震[3] 姜桢[1] 陈灏珠[2] 

机构地区：[1]复旦大学附属中山医院麻醉科,上海200032 [2]复旦大学附属中山医院心内科,上海200032 [3]复旦大学上海医学院病理学系

出　　处：《中华心血管病杂志》2008年第6期551-555,共5页Chinese Journal of Cardiology

基　　金：上海市心血管临床医学中心资助项目（ZX02806）

摘　　要：目的探讨过表达内皮素1（ET-1）对体外培养的大鼠肺细小动脉平滑肌细胞（RPMC）凋亡的影响及其作用机制。方法采用组织灌注法体外分离和培养原代RPMC，经免疫荧光法和电镜鉴定后，脂质体法瞬时转染ET-1基因，用流式细胞技术检测细胞周期和凋亡，免疫印迹法检测丝／苏氨酸蛋白激酶（Akt／PKB）的磷酸化水平和剪切的半胱天冬酶-3（Caspase-3）的表达。结果成功分离和培养出原代RPMC，实时荧光定量逆转录-聚合酶链反应检测显示，RPMC中ETA受体mRNA表达水平明显高于ETB受体。瞬时转染ET-1基因48h后，流式细胞仪结果显示，与空载相比，RPMC的凋亡明显下降。免疫印迹法检测显示，过表达ET-1后，Akt／PKB的磷酸化水平升高，剪切的Caspase-3的表达水平下降。结论过表达ET-1可能主要通过ETA受体，并经Akt／PKB-Caspase-3信号通路抑制RPMC的凋亡，从而在肺动脉高压的血管重塑过程中发挥重要作用。Objective To investigate the effects of endothehn-1 （ET-1） overexpression on apeptosis of the rat pulmonary arterial microvascular smooth muscle cells （ RPMC ） in vitro. Methods Primary RPMC obtained from the pulmonary artery and lung microvasculature were identified by staining and electron microscope technique. The RPMC was transient transfected with the pMEXneo-ET1 and pCDNAS-FRT-TO-ET1-3＇UTR plasmids as well as the empty vector respectively via lipefectamine. Flow cytometry was used to assess the cell cycle and apeptosis of RPMC. Akt and Caspase-3 expressions were detected by Western blot and real time RT-PCR. Results The mRNA of ETA expression was significantly higher than that of ETB receptor in primary RPMC. Flow cytometry analysis revealed significantly reduced apoptosis in ET-1 transfected RPMC compared to that in vehicle transfected RPMC. Overexpression of ET-1 in RPMC also significantly increased the phosphorylation of Akt and reduced the cleaved Caspase-3 expression. Conclusions Overexpression of the ET-1 inhibited RPMC apeptosis and activated Akt/PKB-Caspase-3 signaling pathway, which might be responsible for ET-1 induced the pulmonary microvascular arteries remodeling.

关 键 词：高血压 肺性 肌细胞 平滑肌 内皮缩血管肽1 细胞凋亡 

分 类 号：R686[医药卫生—骨科学]