RhoA-Rho激酶信号通路在血管紧张素Ⅱ刺激心肌成 纤维细胞增殖及.docVIP

RhoA/Rho激酶信号通路在血管紧张素Ⅱ刺激心肌成 纤维细胞增殖及 　 作者：汪祥海， 伍卫， 杨军， 方昶， 耿登峰， 黄至斌 【摘要】 　　【目的】 探讨RhoA/Rho激酶信号通路在血管紧张素Ⅱ（angiotensinⅡ，AngⅡ）刺激心肌成纤维细胞（ cardiac fibroblasts，CFBs）增殖和胶原合成中的作用。【方法】 采用胰酶消化、差速贴壁法培养新生SD 大鼠CFBs,并用AngⅡ诱导CFBs增殖和胶原合成。采用四氮唑盐比色法测定细胞增殖,羟脯氨酸法测定CFBs胶原含量,RT-PCR检测RhoA/Rho激酶mRNA的表达，Western blot检测肌球蛋白结合亚基磷酸化（phosphorylation of myosin-binding subunit,MBS-P）表达作为Rho激酶功能活化的标志。【结果】 AngⅡ(10-7mol/L )刺激48 h可诱导新生SD大鼠CFBs的Rho激酶活化（Ｐlt; 0.01），上调RhoA、Rho激酶mRNA表达(Ｐlt;0.05, Ｐlt; 0.05)；Rho激酶特异性抑制剂Hydroxyfasudil (H4413)对AngⅡ刺激的CFBs增殖与胶原合成具有明显的抑制作用(Ｐlt; 0.05, Ｐlt; 0.05)。【结论】 RhoA/Rho激酶信通路可能在调控AngⅡ刺激CFBs增殖和胶原合成中发挥重要作用。 【关键词】 心肌成纤维细胞； Rho激酶； 血管紧张素Ⅱ Abstract：【Objective】 To investigate the role of RhoA/Rho-kinase signal pathway in cardiac fibroblasts(CFBs) proliferation and collagen synthesis induced by angiotensin Ⅱ(AngⅡ). 【Methods】 CFBs of neonatal Sprague-Dawley (SD) rats were isolated with the method of trypsin digestion and differential anchoring velocity. Stimulation of the CFBs with AngⅡinduced fibrosis model. Proliferation of CFBs was observed by MTT coloricmetric assay. Synthesis of collagen was observed by the hydroxyp roline. The expression of RhoA and Rho-kinase mRNA was examined using semi-quantitative RT-PCR analysis. The extent of phosphorylation of myosin-binding subunit (MBS-P)of myosin phosphatase was quantified by Western blot analysis, and it was used to evaluate the activity of Rho-kinase. 【Results】 Stimulation of neonatal SD rats CFBs with AngⅡ (10-7 mol/L ) significantly activated Rho-kinase(Ｐlt; 0.01), and significantly increased the expression of RhoA and Rho-kinase mRNA(Ｐlt; 0.05, Ｐlt; 0.05). Pretreatment of CFBs with a Rho-kinase inhibitor, hydroxyfasudil (H4413), effectively inhibited AngⅡ-induced CFBs proliferation and collagen synthesis(Ｐlt; 0.05, Ｐlt; 0.05). 【Conclusion】 RhoA/Rho-kinase signal pathway maybe one of the most important signal transducer for AngⅡ-induced CFBs proliferation and collagen synthesis in neonatal SD rats. Key words: cardiac fibroblasts;