专题研究(Monographic Study)

ROCKI/II在转化生长因子β1诱导的主动脉平滑肌细胞表型转化中的作用

Published at: 2017年第26卷第12期

汪海波 1 , 高旭辉 1 , 朱健 1 , 郗二平 1 , 张瑜 1 , 王正 1 , 刘子豪 1 , 谢彪 1 , 朱水波 1
1 中国人民解放军广州军区武汉总医院 心胸外科,湖北 武汉 430070
通讯作者 水波 朱 Email: zhutian126@163.com
DOI: 10.3978/j.issn.1005-6947.2017.12.010
基金:
湖北省武汉市科技局应用基础研究计划基金资助项目 215060101010053

摘要

目的:探讨ROCKI/II在转化生长因子β1(TGF-β1)诱导的人主动脉平滑肌细胞(HA-VSMC)表型转化中的作用。方法:将HA-VSMC分别转染ROCKI和ROCKII的siRNA后荧光显微镜观察转染情况,并用Western blot方法检测不同处理的HA-VSMC(ROCKI siRNA转染、ROCKII siRNA转染、+TGF-β1、ROCKI siRNA转染+TGF-β1、ROCKII siRNA转染+TGF-β1)中ROCKI和ROCKII蛋白的表达;分别用Western blot和RT-PCR方法检测不同处理的HA-VSMC(+TGF-β1、ROCKI siRNA转染+TGF-β1、ROCKII siRNA转染+TGF-β1、ROCK非特异性抑制剂Y-27632预处理+TGF-β1)中细胞收缩表型标志物α-平滑肌肌动蛋白(α-SMA)、平滑肌22α(SM22α)与合成表型标志物骨桥蛋白(OPN)的蛋白与mRNA表达,均以无处理的HA-VSMC为空白对照。结果:免疫荧光观察与Western blot检测表明两种siRNA均成功转染;TGF-β1处理后,HA-VSMC中ROCKI蛋白表达明显升高(P<0.05),但ROCKII蛋白表达无明显变化(P>0.05),ROCKI siRNA转染后TGF-β1上调ROCKI的作用被明显抑制(P<0.05)。与空白对照组HA-VSMC比较,TGF-β1处理后的HA-VSMC中α-SMA、SM22α的蛋白和mRNA表达明显降低,而OPN蛋白与mRNA表达明显升高(均P<0.05),ROCKI siRNA转染或Y-27632预处理后,TGF-β1的上述作用均明显减弱(均P<0.05),ROCKII siRNA转染对TGF-β1的上述作用均无明显影响(均P>0.05)。结论:TGF-β1可诱导HA-VSMC由收缩表型向合成型表型转化,ROCKI表达的升高可能在这一转化中起主要作用。


Effects of ROCKI/II on phenotype switch in aortic vascular smooth muscle cells induced by TGF-β1

Abstract

Objective: To investigate the actions of ROCKI/II in phenotypic transformation of human aortic vascular smooth muscle cells (HA-VSMCs) induced by transforming growth factor β1 (TGF-β1). Methods: HA-VSMCs were respectively transfected with ROCKI and ROCKII, and the transfection results were observed by fluorescence microscope. The ROCKI and ROCKII protein expressions in HA-VSMCs with different treatments (ROCKI siRNA transfection, ROCKII siRNA transfection, +TGF-β1, ROCKI siRNAtransfection+TGF-β1, and ROCKII siRNAtransfection+TGF-β1) were determined by Western blot analysis. The protein and mRNA expressions of the contractile phenotype maker α-smooth muscle actin (α-SMA) and smooth muscle 22α (SM22α) and synthetic phenotype marker osteopontin (OPN) in HA-VSMCs with different treatments (+TGF-β1, ROCKI siRNA transfection+TGF-β1, ROCKII siRNA transfection+TGF-β1, and pretreatment of ROCK non-specificity Y-27632+TGF-β1) were determined by Western blot analysis and RT-PCR method, respectively. Untreated HA-VSMCs were used as blank control. Results: Both siRNAs were successfully transfected as evidenced by fluorescence observation and Western blot analysis. In HA-VSMCs after TGF-β1 treatment, the ROCKI protein expression level was significantly up-regulated (P<0.05), but the ROCKII protein expression level did not significantly change (P>0.05), while the ROCKI increasing effect of TGF-β1 was significantly inhibited by ROCKI siRNA transfection (P<0.05). In HA-VSMCs after TGF-β1 treatment, the protein and mRNA expressions of α-SMA and SM22α were decreased and those of OPN were increased significantly (all P<0.05), and these effects were significantly suppressed by ROCKI siRNA transfection or Y-27632 pretreatment (all P<0.05), but were not affected by ROCKII siRNA transfection (all P>0.05). Conclusion: TGF-β1 can induce the transformation of HA-VSMCs from contractile phenotype to synthetic phenotype, which may be associated with the up-regulation of ROCKI expression.


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引用

引用本文: 海波 汪, 旭辉 高, 健 朱, 二平 郗, 瑜 张, 正 王, 子豪 刘, 彪 谢, 水波 朱. ROCKI/II在转化生长因子β1诱导的主动脉平滑肌细胞表型转化中的作用[J]. 中国普通外科杂志, 2017, 26(12): 1568-1574.
Cite this article as: WANG Haibo, GAO Xuhui, ZHU Jian, XI Erping, ZHANG Yu, WANG Zheng, LIU Zihao, XIE Biao, ZHU Shuibo . Effects of ROCKI/II on phenotype switch in aortic vascular smooth muscle cells induced by TGF-β1[J]. Chin J Gen Surg, 2017, 26(12): 1568-1574.