ShRNA介导的Smad4基因沉默对C2C12成肌细胞纤维化的影响  

Effects ShRNA-mediated Gene Silencing Smad4 on the Myofibrogenesis of C2C12 Myoblasts

作　　者：陈始秋[1] 陈疾忤[1] 陈世益[1] 李宏云[1] 尚西亮[1] 蒋佳[1] 

机构地区：[1]复旦大学附属华山医院运动医学科,上海200040

出　　处：《中国运动医学杂志》2011年第12期1100-1105,共6页Chinese Journal of Sports Medicine

基　　金：国家自然科学基金项目(NSFC30800543)

摘　　要：目的:研究慢病毒介导的Smad4-shRNA对TGF-β1诱导的纤维化进程的影响。方法:(1)设计并选择抑制效能最高的Smad4-shRNA,包装生产慢病毒,转染C2C12成肌细胞,并检测转染后细胞的Smad4表达;(2)根据TGF-β1诱导C2C12成肌细胞向成肌纤维细胞分化的模型,以慢病毒介导的Smad4-shRNA转染细胞,将不同处理的细胞分为A、B、C、D四组,分别为C2C12细胞组、TGF-β1诱导组、C2C12细胞转染组和TGF-β1诱导后转染组。通过荧光Realtime-PCR和Westerblot检测各组collagen I和α-SMA表达水平。结果:(1)慢病毒介导Smad4-shRNA1转染C2C12细胞后,其Smad4 mRNA和蛋白表达显著低于未转染组(P<0.05);(2)TGF-β1诱导组α-SMA和Collagen I mRNA及蛋白表达均显著高于C2C12细胞组(P<0.05);(3)TGF-β1诱导后转染组α-SMA与collagen I mRNA及蛋白表达显著低于TGF-β1诱导组(P<0.05),与C2C12细胞组和C2C12细胞转染组相比则无明显差异(P>0.05)。结论:降低Smad4表达能有效抑制成肌细胞及受TGF-β1诱导成肌纤维细胞的纤维化过程,提示Smad4可能成为拮抗骨骼肌纤维化的靶点。Objective To study the effect of Smad4-shRNA on the myofibrosis process.Methods（1）The most efficient silencing Smad4-shRNA was designed and used to produce lentivirus-Smad4 shRNA to infect C2C12 myoblasts.The Smad4 expression was examined.（2）According to the model that TGF-β1 induced the myofibrosis process of the C2C12 myoblasts,the lentivirus mediated cells were divided into four groups：group A,B,C,and D,respectively presenting the normal C2C12 cells group,the TGF-β1 induced C2C12 cells group,the lentivirus-mediated cells group and the TGF-β1 induced lentivirus-mediated cells group.Results（1）The mRNA and protein expression of Smad4 showed a lower level in Smad4-shRNA1 Lentivirus-mediated C2C12 cells（P 0.05）;（2）The collagen I and α-SMA expression in group B were higher than in group A（P 0.05）;（3）The collagen I and α-SMA expression in group D decreased obviously as compared with that in group B（P 0.05）,without significant difference between group A and group C（P 0.05）.Conclusion Smad4 down-regulation could effectively suppress myofibrosis induced by TGF-β1,and Smad4 inhibition was a potential method for anti-fibrosis of the injured skeletal muscle.

关 键 词：骨骼肌损伤 纤维化 SMAD4 TGF-Β1 C2C12成肌细胞 

分 类 号：R873[医药卫生—运动医学]