内皮素-1经蛋白激酶B/雷帕霉素靶蛋白信号通路诱导大鼠肺细小动脉平滑肌细胞肥大

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)增殖/肥大的影响及其作用机制研究.方法 采用组织灌注法体外分离培养RPMC,脂质体介导法瞬时转染ET-1基因,经实时荧光定量逆转录-PCR和免疫印迹法鉴定后,用流式细胞技术检测细胞周期和细胞体积的变化,用免疫印迹法和细胞免疫荧光法检测α-平滑肌肌动蛋白(α-SMA)的表达,同时检测细胞总蛋白/总DNA比值的变化,并用免疫印迹法检测蛋白激酶B(Akt/PKB)、雷帕霉素靶蛋白(mTOR)和细胞外信号调节激酶(ERK1/2)的磷酸化水平.采用SPSS 11.0统计软件处理数据,各组间比较均采用t检验.结果 成功分离和培养RPMC并瞬时转染ET-1基因.与空载相比,转染48 h后RPMC细胞周期分布和细胞增殖指数未见明显差异,但转染72 h后检测到α-SMA合成功能增强,分别为空载转染组的1.3倍和1.2倍,细胞总蛋白/总DNA比也增高,分别为空载转染组的1.6倍和1.5倍.流式细胞术检测显示,与空载相比,转染72 h后RPMC细胞体积增大,分别为空载转染组的1.1倍和1.2倍.免疫印迹法检测显示,转染48 h后,Akt/PKB和mTOR的磷酸化水平升高,ERK1/2的磷酸化水平降低.结论 过度表达ET-1可能经Akt/PKB-mTOR信号通路诱导RPMC肥大,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 endothelin-1(ET-1)overexpression on hypertrophy of the rat pulmonary arterial microvascular smooth muscle cells(RPMCs)in vitro.Methods Lung tissue perfusion method was used to obtain the primary RPMCs,and then the cells were transiently transfected with ET-1 plasmids or empty vectors via Lipofectamine.Flow cytometry was used to determine the distributions of the cell cycle and cell size of RPMCs.Immunofluorescence staining and Western blotting were used to determine the level of α-smooth muscle actin.The ratio of the protein/DNA of the transfected RPMCs was also calculated.Western blotting was used to examine the levels of phosphorylations of protein kinase B/Akt,mTOR,and ERK1/2.Results Primary RPMCs were isolated successfully.Overexpression of ET-1 resulted in a significant increase in total protein synthesis,expression of α-SMA,as well as increased cell size.Western blotting results showed that overepression of ET-1 resulted in increased phosphorylation of Akt,mTOR and a decreased phosphorylation of ERK1/2 in RPMC.Conclusions ET-1 overexpression induces RPMC hypertrophy and activation of Akt/PKB-mTOR pathway may be involved in the mechanism,with important implications for the pathogenesis of vascular remodeling in pulmonary arterial hypertension.