Smad7在肌腱粘连组织中的表达及对肌腱成纤维细胞纤维化的作用

Expression of Smad7 in tendon adhesion tissue and its role in fibrosis of tendon derived fibroblasts

[目的]通过观察人肌腱粘连组织中Smad7的表达情况,并在TGF-β1诱导的肌腱成纤维细胞(TDFs))中沉默或过表达Smad7,观察细胞纤维化相关指标的变化,讨论Smad7在肌腱粘连中的作用及调控机制。[方法]收集肌腱粘连组织及正常肌腱腱周组织,Western blotting检测Smad7蛋白含量。设计并合成针对Smad7基因编码区的siRNA,同时构建Smad7基因表达载体,经脂质体转染至TDFs细胞,Real Time-PCR检测胞内Smad7基因含量,Western-blot检测细胞中Smad7蛋白表达;使用TGF-β1诱导转染后TDFs细胞,CCK-8检测细胞增殖活性变化,Western blotting检测纤维化相关的蛋白指标。[结果]与正常腱周组织比较,肌腱粘连组织中Smad7蛋白减少。siRNA及Smad7基因过表达载体转染TDFs,可以明显抑制或者上调细胞内Smad7基因含量及蛋白表达,与未转染组比较,差异均有统计学意义(P<0.05);TGF-β1诱导的TDFs细胞,可以明显增强细胞增殖活性(P<0.01,vs未诱导组),siRNA转染后,TGF-β1诱导TDFs增殖活性和纤维化蛋白指标上升的趋势进一步增强,Smad7基因表达载体转染则使得TGF-β1诱导TDFs增殖活性和纤维化蛋白指标增强的趋势得到明显抑制,基因干预且诱导组与只诱导组细胞活性和蛋白含量相比较,差异均有统计学意义(P<0.05)。[结论]肌腱粘连组织中Smad7蛋白减少,提示肌腱粘连发生过程中Smad7表达受抑制;Smad7基因过表达可以抑制TGF-β1诱导TDFs细胞纤维化。

[Objective] To investigate the expression Smad7 protein in tendon adhesion tissue,and to analyze the effects of Smad7 on tendon fibrosis. [Methods] Expression of Smad7 in tendon adhesion tissues and normal tendon tissues were detected by Western blotting method. siRNA and pc NDA were designed and produced for Smad7,siRNA- Smad7 and pc DNA3-Smad7 were transfected into cultured tendon derived fibroblasts（ TDFs） by lipofectamine. Transfected TDFs were then induced by TGF- β1. The proliferation activity of the cells were measured by the CCK- 8. The expression of fibrosis related proteins in TDFs were detected by Western blotting. [Results] The expression of Smad7 in tendon adhesion tissues decreased more significantly than in the normal tendon tissues（ P〈0. 01）. The gene expression and protein of smad7 were inhibited in siRNA-Smad7 infected TDFs,and both were increased in pc DNA3- Smad7 infected TDFs,showing significant differences compared with the results from non- infected group（ P〈0. 05）. Cell proliferation and the fibrosis related proteins were increased in TGF- β1 induced TDFs,and higher in TGF- β1 induced TDFs after being transfected by siRNA- Smad7. However,the expression was inhibited in pc DNA3- Smad7 group,with significant difference（ P〈0. 05）. [Conclusion] Expression of Smad7 protein may be inhibited in tendon adhesion tissues,and TGF- β1 induced TDFs fibrosis can be significantly inhibited by overexpression of Smad7.