TGF-β1-Erk-CTGF信号通路在体外骨骼肌肌源性干细胞分化作用中的研究

The role of TGF-β1-Erk-CTGF signal pathway in the process of skeletal muscle-derived stem cell differentiation： an in vitro study

目的 探讨转化生长因子β1 （TGF-β1）诱导骨骼肌肌源性干细胞（muscle-derived stem cells,MDSCs）向肌成纤维母细胞分化过程中的信号通路.方法 采用差速贴壁法分离出大鼠骨骼肌肌源性干细胞,培养基中加入TGF-β1和细胞外信号调节激酶（extracellular signal-regulated kinase,Erk）特异性拮抗剂PD98059.将体外培养的细胞分为三组：对照组（A组）,10 ng/ml TGF-β1组（B组）,10 ng/ml TGF-β1＋ 80μM PD98059组（C组）.利用免疫细胞化学染色方法检测细胞表型的变化,RT-PCR、Western-blot检测细胞中CTGF和COL Ⅰ表达和合成水平.结果 在体外,TGF-β1能诱导MDSCs高表达α-SMA、CTGF和COLⅠ,抑制MDSCs合成Sac-1;Erk特异性拮抗剂PD98059能抑制TGF-β1的作用,显著降低细胞CTGF和COLⅠ mRNA的表达水平,减低CTGF和COLⅠ合成,增加Sac-1的合成.结论 TGF-31能在体外诱导骨骼肌MDSCs向肌成纤维母细胞分化,TGF-β1-Erk-CTGF信号传导通路参与细胞分化过程.

Objective To investigate the signal pathway of TGF-β1 in the process of muscle-derived stem cell （MDSCs） differentiation towards myofibroblasts.Methods MDSCs were isolated from rat skeletal muscles via differential adhesion method.TGF-β1 and PD98059,a specific antagonist to extracellular signal-regulated kinase （Erk） were added in the culture medium.Three cell culture groups were set up：group A,control; group B,10 ng/ml TGF-β1; group C,10 ng/ml TGF-β1 ＋ 80 μM PD98059.Immunocytochemistry was employed to determine cell phenotypes.RT-PCR and Western Blot were used to detect the expression of Sca-1,α-SMA,CTGF and COL Ⅰ in isolated cells under the stimulation of TGF-β1 alone or combination of TGF-β1 and PD98059.Results In vitro,TGF-β1 upregulated the expression of α-SMA,CTGF and COL Ⅰ and inhibited the synthesis of Sca-1 in MDSCs.However,PD98059 counteracted TGF-β1,resulting in decreased mRNA expression and protein synthesis of CTGF and COL Ⅰ and increased protein synthesis of Sca-1.Conclusion TGF-β1 induces the in vitro differentiation of skeletal MDSCs towards myofibroblasts.TGF-β1-Erk-CTGF signal pathway plays an important role in the cell differentiation process.