miR-146a调控脂肪间充质干细胞成骨分化的机制

Mechanism by which miR-146a regulates osteogenic differentiation of adipose derived mesenchymal stem cells

背景:既往研究表明,miR-146a能影响骨髓间充质干细胞成骨分化,但其在脂肪间充质干细胞成骨分化中的调节功能尚未阐明。目的:探究miR-146a对小鼠脂肪间充质干细胞成骨分化的调控作用。方法:获取雄性C57BL/6小鼠脂肪间充质干细胞,采用倒置显微镜观察细胞形态,流式细胞学方法检测第3代细胞CD29、CD44、CD45的表达,然后用mi R-NC、mi R-146a mimics和mi R-146a inhibitor转染脂肪间充质干细胞并进行成骨诱导分化,成骨诱导第6天,采用碱性磷酸酶染色观察钙化程度,RT-PCR和Western blot检测成骨表型标志物的表达;成骨诱导第12天,采用茜素红染色鉴定细胞表面矿化基质的产生。结果与结论:①培养的细胞为长梭形,呈成纤维样生长,第3代细胞CD29、CD44高表达,CD45低表达;②随着成骨诱导时间的延长,小鼠脂肪间充质干细胞miR-146a的表达逐渐降低(P<0.05);③与miR-NC组相比,碱性磷酸酶显色及其活性在miR-146a mimic组显著减弱,而在miR-146a inhibitor组显著增强(P<0.05);④与miR-NC组相比,ALP、Runx2、Akt、PI3K的m RNA和蛋白表达及p-Akt与p-PI3K的蛋白表达在miR-146a mimic组显著降低,而在miR-146a inhibitor组显著升高(P<0.05);⑤与miR-NC组相比,茜素红染色发现miR-146a mimic组细胞的钙化程度显著降低(P<0.05),而miR-146a inhibitor组的钙化程度显著升高(P<0.05);⑥上述结果表明,miR-146a能够通过PI3K/AKT信号通路负向调控脂肪间充质干细胞向成骨细胞分化。

BACKGROUND:Previous studies have shown that miR-146a can affect the osteogenic differentiation of bone marrow mesenchymal stem cells,but its regulatory function in adipose derived mesenchymal stem cells has not been elucidated.OBJECTIVE:To investigate the regulatory role of miR-146a in the osteogenic differentiation of mouse adipose derived mesenchymal stem cells.METHODS:Adipose derived mesenchymal stem cells were obtained from male C57BL/6 mice.The cell morphology was observed by inverted microscope,and the expression of CD29,CD44 and CD45 of passage 3 cells was detected by flow cytometry.miR-NC,miR-146a mimics and miR-146a inhibitor were transfected into adipose derived mesenchymal stem cells for osteogenic differentiation.On day 6 of osteogenic induction,alkaline phosphatase staining was used to observe the degree of calcification.RT-PCR and western blot assay were used to detect the expression of osteogenic phenotype markers.On day 12 of osteogenic induction,alizarin red staining was used to identify the formation of mineralized matrix on the cell surface.RESULTS AND CONCLUSION:(1)The cultured cells were spindle shaped and fibroblast like.The expression of CD29 and CD44 was high and that of CD45 was low.(2)The expression of miR-146 a in mouse adipose derived mesenchymal stem cells decreased with the prolongation of osteogenic induction time(P<0.05).(3)Compared with miR-NC group,the amount and activity of alkaline phosphatase precipitates in miR-146a mimic group were significantly lower than those in miR-146a inhibitor group(P<0.05).(4)Compared with miR-NC group,the mRNA and protein expressions of ALP,Runx2,Akt,p-Akt,PI3K and p-PI3K were significantly decreased in miR-146a mimic group,but significantly increased in miR-146a inhibitor group(P<0.05).(5)Compared with miR-NC group,alizarin red staining showed that the degree of calcification in miR-146a mimic group was significantly lower(P<0.05),while that in miR-146a inhibitor group was significantly higher(P<0.05).(6)These results suggest that miR-146a can negatively regulate the differentiation of adipose derived mesenchymal stem cells into osteoblasts through PI3K/AKT signaling pathway.