Smad6信号干扰对MSCs骨向分化的影响

The effect of Smad6 RNA interference on BMP-2 induced osteogenic differentiation of mesenchymal stem cells

目的研究Smad6信号干扰对骨形态发生蛋白2(BMP-2)诱导的骨髓间充质干细胞(MSCs)骨向分化的促进效应。方法培养小鼠MSCs,用BMP-2诱导骨向分化。细胞分为3组:A组细胞用携带绿色荧光蛋白(GFP)的Smad6重组RNA干扰载体转染;B组细胞用空白载体转染;C组细胞作为对照。结果病毒转染后GFP在MSCs中有效表达,病毒转染效率达98.5%。与B组比较,Smad6 RNA干扰显著提高了A组细胞ALP活性和骨钙素水平(P<0.01),而C组ALP活性和骨钙素水平均显著低于其他2组(P<0.01)。茜素红染色显示,A组矿化结节数目显著多于B组(P<0.05),而C组无矿化结节形成。结论 Smad6 RNA干扰可有效促进BMP-2诱导的MSCs骨向分化,该研究为骨组织工程中骨缺损修复提供了一个极具价值的手段。

Objective To investigate the effect of Smad6 mRNA interference on 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 BMP-2 induced osteogenic differentiation. The cells were divided into 3 groups： the cells in group A were transfected with recombinant Smad6 RNA interference vector,which was labeled with green fluorescent protein （ GFP）, and the ceils in group B were transfected with control vector, and the cells in group C served as controls. The activity of alkaline phosphonate （ALP） and levels of osteocalcin were detected at five days after transfection by ALP staining and radioimmunoassay, respectively. The formation of mineralization nodus was also examined by alizarin red staining. Results The GFP was obviously expressed in MSCs after viral transfection, and viral transfection efficiency reached 98.5%. As compared with group B, Smad6 RNA interference increased significantly ALP activity and osteocalcin levels in group A （ P 〈 O. 01 ）, however, both ALP activity and osteocalcin levels in group C were significantly lower than those in the other two groups（ P 〈 0.01 ）. The results of alizarin red staining showed that the counts of mineralization nodus in group A were significantly more than those in group B （ P 〈 O. 05）, but no mineralization nodus was found in group C. Conclusion Smad6 mRNA interference, can promote effectively BMP-2 induced osteogenic differentiation of MSCs,which may be a valuable method for bone regeneration for bone defect in bone tissue engineering.