Smad6信号干扰对MSCs骨向分化中Smad5及Smurf1基因表达的影响

The influence of Smad6 RNA interference on gene expression of Smad5 and Smurf1 during osteogenic differentiation of MSCs

目的研究在骨形态发生蛋白2(BMP-2)诱导的骨髓间充质干细胞(MSCs)骨向分化中Smad6信号干扰对Smad5、Smurf1基因表达的影响。方法培养小鼠骨髓MSCs,并分为3组:A组为对照细胞;B组细胞用携带绿色荧光蛋白(GFP)的空白载体病毒转染+BMP-2骨向诱导;C组细胞用Smad6重组RNA干扰载体转染+BMP-2诱导。结果病毒转染后GFP在MSCs中有效表达,病毒转染效率接近100%。与A组比较,BMP-2诱导24h显著提高了B组Smurf1 mRNA水平(P<0.01);而Smad6信号干扰在两个时间点则进一步提高了C组Smurf1 mRNA水平,并显著高于B组(P<0.01)。C组磷酸化的Smad5(pSmad5)和Smurf1水平在两个时间点也显著高于B组(P<0.01),而Smad5蛋白水平则未受影响。结论在BMP-2诱导的MSCs骨向分化中,Smad6 RNA干扰可促进Smad5磷酸化,并引起Smurf1基因表达代偿性增高。

Objective To investigate the influence of Smad6 RNA interference on gene expression of Smad5 and smad ubiguitin regulatory factor-1（Smurf1） during bone morphogenetic protein 2（BMP-2） induced osteogenic differentiation of bone marrow mesenchymal stem cells（MSCs）.Methods The bone marrow MSCs of mice were cultured and underwent osteogenic differentiation induced by BMP-2.The cells were divided into 3 groups：group A（control cells）,group B（control vector plus BMP-2 induction）,and group C（Smad6 recombinant RNA interference vector plus BMP-2 induction）.The expression of GFP was observed under fluorescent microscope.At 6 and 24 hours after ostegenic induction,mRNA levels of Smurf1,protein levels of Smad5 and Smurf1 were detected by SyberGreen Real-time PCR and Western-blot respectively.Results GFP was effectively expressed in MSCs and high transfection efficiency（about 100%） was obtained after viral transfection.BMP-2 induction significantly increased mRNA levels of Smurf1 in group B at 24 hours after induction,as compared with those in group A（P0.01）,however,Smad6 RNA interference increased mRNA levels of Smurf1 further in group C at both time points,as compared with those in group B（P0.01）.The protein levels of Smad5 and Smurf1 were also significantly increased in group C,as compared with those in group B at both time points,but the protein levels of Smad5 were not affected.Conclusion Smad6 mRNA interference can effectively promote phosphorylation of Smad5 and result in compensatory increase of gene expression of Smurf 1 during BMP-2 induced osteogenic differentiation of MSCs.