TGF-β_1介导的RhoA/ROCK通路在大鼠肺肌成纤维细胞分化中的调节作用  

Regulation of rat pulmonary fibroblasts differentiation into myofibroblasts though RhoA /ROCK pathway mediated by TGF-β_1

作　　者：马文东[1] 袁媛[1] 杨奕[2] 徐洪[1] 邓海静[1] 于婉莹[1] 孙月[1] 魏中秋[3] 杨方[1] 

机构地区：[1]河北联合大学医学实验研究中心,河北唐山063000 [2]河北联合大学教务处,河北唐山063000 [3]河北联合大学病理学教研室,河北唐山063000

出　　处：《中国病理生理杂志》2013年第10期1758-1763,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81072254);人事部留学人员科技活动项目(No.国人厅发[2006]164号);河北省自然科学基金资助项目(No.C2011401024)

摘　　要：目的:探讨转化生长因子β1(TGF-β1)介导的RhoA/Rho相关卷曲螺旋形成蛋白激酶(ROCK)通路在调控肺成纤维细胞向肌成纤维细胞分化过程中的作用。方法:胰酶消化法获得原代培养的肺成纤维细胞,进行以下实验:(1)观察TGF-β1诱导肺成纤维细胞不同时间后p-RhoA、ROCK、磷酸化肌球蛋白磷酸酶靶亚基(pMBS)、血清应答因子(SRF)、α-平滑肌肌动蛋白(α-SMA)、I型和III型胶原蛋白表达变化;(2)将细胞分为对照组、TGF-β1诱导分化组和Y-27632(ROCK通路阻滞剂)干预组,免疫细胞化学染色与Western blotting法分别观察与检测ROCK、p-MBS、SRF、α-SMA、I型和III型胶原蛋白在细胞内的定位分布与表达。结果:(1)经TGF-β1诱导24 h后,大鼠肺成纤维细胞胞体内出现大量平行或交叉排列的α-SMA抗体标记的肌丝。随着TGF-β1诱导刺激时间的延长,p-RhoA、ROCK、p-MBS、SRF、α-SMA、I型和III型胶原蛋白表达逐渐增高,其中RhoA/ROCK信号(p-RhoA、ROCK、p-MBS)、SRF和α-SMA蛋白分别在诱导后6 h、12 h和24 h达到高峰。I型胶原和III型胶原蛋白表达均在24 h达高峰,分别为诱导前的2.19和3.04倍(P<0.05)。(2)与TGF-β1诱导分化组比较,当给予Y-27632干预后,在相应时点ROCK、p-MBS、SRF、α-SMA、I型和III型胶原蛋白表达均明显降低,差异有统计学意义(P<0.05)。结论:TGF-β1通过ROCK信号转导通路的活化,刺激了大鼠肺成纤维细胞向肌成纤维细胞分化,进而促进了胶原蛋白的合成,可能在(矽)肺纤维化形成过程中发挥着重要作用。AIM：To investigate the regulatory effect of RhoA/Rho-associated coiled-coil-forming protein kinase （ROCK） pathway mediated by transforming growth factor β1 （TGF-β1） on the differentiation of pulmonary fibroblasts into myofibroblasts. METHODS：Primarily cultured fibroblasts were obtained by trypsin digestion from the lung of neonatal rats. The fibroblasts were stimulated with TGF-β1 for different durations and were divided into control group, TGF-β1 induction group and Y-27632 treatment group. The distribution and expression of p-RhoA, ROCK, phosphorylated myosin binding subunit of myosin light chain phosphatase （p-MBS）, serum response factor （SRF）, α-smooth muscle actin （α-SMA）,type I collagen and type Ⅲ collagen in the cells were detected by the methods of immunocytochemistry and Western blotting. RESULTS：A lot of parallel and cross arranged filaments labeled by α-SMA antibody appeared in the cells after TGF-β1 stimulation. The cultured cells stimulated with TGF-β1 were all myofibroblasts at 24 h determined by immunocytochemistry. The expression levels of p-RhoA, ROCK, p-MBS, SRF, α-SMA and type I and type III collagens were increased gradually with the extension of TGF-β1 stimulation time. The expression of RhoA/ROCK signaling protein in the cells stimulated with TGF-β1 （peaking at 6 h of exposure） was 2.96 folds higher as compared with the non-stimulated cells. The expression of SRF protein （peaking at 12 h of TGF-β1 exposure） was 4.55 folds higher as compared with the non-sti-mulated cells. The expression levels of α-SMA and type I and type III collagens （peaking at 24 h of TGF-β1 exposure） were 4.06 folds, 2.19 folds and 3.04 folds higher as compared with the non-stimulated cells, respectively. Compared with TGF-β1 induction group, the protein expression levels of ROCK, p-MBS, SRF, α-SMA and type I and type III collagens were significantly decreased at the corresponding time points in Y-27632 treatment group. CONCLUSION：TGF-β1 induces the differenti

关 键 词：转化生长因子Β Rho相关卷曲螺旋形成蛋白激酶 肌成纤维细胞 肺纤维化 

分 类 号：R135.2[医药卫生—劳动卫生]