【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si RNA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 si RNA转染、ROCKII si RNA转染、+TGF-β1、ROCKI si RNA转染+TGF-β1、ROCKII si RNA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 si RNA转染+TGF-β1、ROCKII si RNA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m RNA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si RNA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 si RNA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m RNA表达明显降低,而OPN蛋白与m RNA表达明显升高(均P<0.05),ROCKI si RNA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 si RNA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。更多还原

【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 si RNA transfection, ROCKII si RNA transfection, +TGF-β1, ROCKI si RNAtransfection+TGF-β1, and ROCKII si RNAtransfection+TGF-β1) were determined by Western blot analysis. The protein and m RNA 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 si RNA transfection+TGF-β1, ROCKII si RNA transfection+TGF-β1, and pretreatment of ROCK non-specificity Y-27632+TGF-β1) were determined by Western blot analysis and RTPCR method, respectively. Untreated HA-VSMCs were used as blank control.Results: Both si RNAs 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 upregulated(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 si RNA transfection(P<0.05). In HAVSMCs after TGF-β1 treatment, the protein and m RNA 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 si RNA transfection or Y-27632 pretreatment(all P<0.05), but were not affected by ROCKII si RNA 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.更多还原