【摘要】 目的探讨miR-224对骨髓间充质干细胞(hBMSCs)成骨分化的影响及其机制。方法体外以成骨诱导培养基培养hBMSCs,采用Western印迹法和碱性磷酸酶(ALP)活性测定试剂盒检测成功分化标志物骨钙素(OCN)、成骨特异性转录因子(Runx)2蛋白和ALP活性。采用脂质体2000转染miR-224 mimics和Smad4 siRNA干扰序列分别上调miR-224和下调Smad4表达,以实时荧光定量聚合酶链反应(qRT-PCR)和Western印迹法检测miR-224和Smad4表达情况,采用荧光素酶报告基因实验检验miR-224与Smad4的靶向关系。结果随hBMSCs诱导分化时间的延长,miR-224表达降低,而Smad4表达升高。上调miR-224后,成骨分化相关指标ALP活性、OCN和Runx2蛋白表达减弱,Smad4蛋白表达下降;荧光素酶实验证实Smad4是miR-224的靶基因,下调其表达后,成骨分化相关指标ALP活性、OCN和Runx2蛋白表达减弱。结论 miR-224可通过靶向Smad4抑制hBMSCs成骨分化。更多还原

【Abstract】 Objective To investigate the effect of miR-224 on osteogenic differentiation of bone marrow mesenchymal stem cells(hBMSCs) and its mechanism.Methods hBMSCs was cultured in vitro by osteogenic induction medium. The OCN, Runx2 proteins and ALP activity of the differentiation markers were detected by Western blot and ALP activity assay kit. miR-224 mimics and Smad4 siRNA interference sequences were transfected by liposome 2000, which up-regulated miR-224 and down-regulated Smad4 expression. The expression of miR-224 and Smad4 were measured by qRT-PCR and Western blot, and the relationship between miR-224 and Smad4 was examined by Luciferase reporter gene assay.Results With the prolongation of hBMSCs induction-differentiation time, the expression of miR-106 a was decreased, while the expression of Smad4 was increased. After up-regulation of miR-224, the activity of ALP, the expression of OCN and Runx2 proteins were cut down, and the expression of Smad4 protein declined. The luciferase test confirmed that Smad4 was the target gene of miR-224, and osteogenic differentiation related indexes ALP activity, OCN and Runx2 proteins were weakened after the down-regulation of Smad4 expression.Conclusions miR-224 inhibits the osteogenic differentiation of bone marrow mesenchymal stem cells by targeting Smad4.更多还原