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
| 作者: |
1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 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: |
朱水波
Email: zhutian126@163.com |
| DOI: | 10.3978/.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。
关键词：
肌，平滑，血管；动脉瘤，夹层；rho相关激酶类；ROCKI/II；表型
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
CorrespondingAuthor:ZHU Shuibo Email: zhutian126@163.com
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.
Keywords：
Muscle
Smooth
Vascular; Aneurysm
Dissecting; rho-Associated Kinases; Phenotype
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.