Objective To investigate the effects and possible mechanism of the Rho/Rock signaling pathway on TGF-β1 -induced of rat ISMCs phenotypic modulation.Methods The rat ISMCs were randomly divided into two groups:blank control group and TGF-β1 stimulation group, in which TGF-β1 of different concentrations (1, 5, 10, 20 and 50 ng/mL) were given for 24 h.Transgelin ( SM22α) and osteopontin ( OPN) mRNA expression was assessed by flu-orescence quantitative real-time polymerase chain reaction ( FQ-RT-PCR) .The derived optimal concentration was ap-plied to stimulate cells, which were collected at different times (6, 12, 24 and 48 h).After 24 h of serum-free DMEM high glucose medium culture, ISMCs were randomly divided into 3 groups: blank control group, TGF-β1 (10ng/mL) stimulation group, TGF-β1 (10 ng/mL)+Fasudil (10, 30 and 50 μmol/L) groups.The expression levels of SM22α, OPN, RhoA, Rock-1 mRNA were assessed with FQ-RT-PCR;and SM22αand OPN expression levels were assessed using Western Blot.The ultrastructure of the cells was observed by transmission electron microscopy.Results TGF-β1 in 10 ng/mL stimulation of ISMCs reached the summit at 24 h.SM22 mRNA and protein expression levels were signifi-cantly increased in TGF-β1 +Fasudil group (P<0.01).Significant reduction in OPN mRNA was observed in TGF-β1+Fasudil group in dose-dependent manners, and reached lowest effects with Fasudil of 30μmol/L (P<0.05).So was the protein expression of OPN according to Western blot.According to FQ-RT-PCR, the mRNA expression of RhoA and Rock-1 and Rock-1 was significantly increased in TGF-β1 group ( P<0.01 ) , and it was significantly lower in TGF-β1 +Fasudil (30 μmol/L) group than TGF-β1 group (P<0.05).Under electron microscope, normal Golgi ISMC hypertrophy, mitochondria and rough endoplasmic reticulum were observed in control group ( ×20 000) , while in-creased intracellular mitochondria was observed in TGF-β1 group, which was reversed by Fasudil.Conclusion TGF-β1 of 10ng/mL stimulation in ISMCs for 24 h can be successfully induced phenotype modulation.Rho/Rock signaling pathway inhibitors Fasudil increases of TGF-β1 -induced SM22αexpression, loweres OPN expression, thus inhibits ISMCs phenotypic modulation.%目的：探讨Rho／Rock信号通路在TGF－β1刺激大鼠颈内动脉平滑肌细胞（ ISMC）表型转化中的作用及可能机制。方法 ISMC随机分为两组：空白对照组和TGF－β1刺激组，以不同浓度（1、5、10、20、50 ng／mL） TGF－β1刺激细胞24 h，用荧光实时定量PCR法检测转凝蛋白（ SM22α）和骨桥蛋白（ OPN） mRNA的表达；再以上述得出的最佳TGF－β1浓度刺激细胞，在不同时间（6、12、24、48 h）收集细胞，采用同样方法检测SM22α、OPN mRNA的表达水平；经无血清DMEM高糖培养基静止培养24 h后，随机分为以下各组：空白对照组、TGF－β1（10 ng／mL）刺激组及TGF－β1（10 ng／mL）＋法舒地尔（Fasudil）（10、30、50μmol／L）组，分别采用FQ－RT－PCR法检测SM22α、OPN、RhoA、Rock－1 mRNA的表达；Western blot法检测SM22α、OPN蛋白表达水平；根据实验结果用透射电镜观察各组细胞超微结构的相应变化。结果10 ng／mL TGF－β1刺激大鼠ISMCs 24 h达顶峰。 TGF－β1＋Fasudil组SM22αmRNA和蛋白表达水平较TGF－β1刺激组高（P＜0．01），且在Fasudil浓度为30μmol／L时达到高峰，随后下降；TGF－β1＋Fasudil组OPN mRNA表达水平较TGF－β1刺激组低，呈浓度依赖性，至30μmol／L达最低（ P＜0．05），后又呈上升趋势。 Western blot结果显示TGF－β1＋Fasudil组的OPN蛋白表达水平较TGF－β1刺激组低，至Fasudil浓度为30μmol／L达最低（ P＜0．05）。同时FQ－RT－PCR结果显示TGF－β1刺激组的RhoA和Rock－1 mRNA表达水平较空白对照组高（P＜0．01），在Fasudil浓度为30μmol／L时TGF－β1＋Fasudil组的RhoA和Rock－1mRNA表达水平较TGF－β1刺激组低（ P＜0．05）。电镜下观察各组细胞显示，空白对照组（×20000） ISMC高尔基体肥大，线粒体及粗面内质网正常，10 ng／mL TGF－β1刺激后细胞内线粒体增多，在上述基础上加入30μmol／L Fasudil后电镜下观察细胞又回到正常成熟结构状态。结论10 ng／mL TGF－β1刺激大鼠ISMC 24 h可成功刺激其表型转化，且该过程可能通过Rho／Rock信号通路实现；Rho／Rock信号通路抑制剂Fasudil可增加TGF－β1刺激的SM22α的表达，下调OPN的表达水平，抑制大鼠ISMCs表型转化。
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