miR-449对骨髓间充质干细胞成骨分化调控的机制研究

The Mechanism of MiR-449 on Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells

目的探究miR-449是否具有调控骨髓间充质干细胞(MSCs)成骨分化的作用。方法分离、培养骨髓MSCs,采用流式细胞仪检测CD44、CD29、CD34、CD45等表面相关标志抗原的表达鉴定骨髓MSCs;将MSCs分为对照组、miR-449a组、miR-449b组及anti-miR-449组,对照组用成骨诱导培养基培养,其余各组分别经miR-449a/b或inhibitors转染后在成骨诱导培养基培养,分别于培养第3、6、9、12天检测各组碱性磷酸酶活性,qRT-PCR检测miR-449a/b对成骨特异性基因Runx2、Osterix表达的影响。结果①第4代间充质干细胞CD44、CD29表达阳性,而CD34和CD45表达阴性,符合骨髓间充质干细胞的特征。②成骨诱导培养3、6、9、12 d后,MSCs细胞碱性磷酸酶活性较上一时间点升高,且呈时间依赖性,差异有统计学意义(P<0.05);MSCs成骨诱导培养3、6、9、12 d后,Runx2、Osterix表达水平逐渐升高。③miR-449a组和miR-449b组AKP活性低于对照组,而anti-miR-449组AKP活性升高,差异有统计学意义(P<0.05);miR-449a组和miR-449b组Runx2、Osterix表达较对照组下调,差异有统计学意义(P<0.05)。结论本次实验成功分离、培养了骨髓MSCs,其具有体外成骨分化能力,而miR-449能抑制骨髓MSCs向成骨分化。

Objective To investigate whether miR-449 can regulate the osteogenic differentiation of bone marrow mesenchymal stem cells(MSCs).Methods Bone marrow MSCs were isolated and cultured.Flow cytometry was used to detect the expression of surface-associated marker antigens such as CD44,CD29,CD34,and CD45.Bone marrow MSCs were identified.The MSCs were divided into control group,miR-449a group,miR-449b group,and anti-miR-449 group,the control group was cultured with osteogenic induction medium,and the remaining groups were cultured in osteogenic induction medium after transfection with miR-449a/b or inhibitors,respectively in culture 3,6,9,and12 d,the alkaline phosphatase activity was detected in each group,and the effect of miR-449a/b on the expression of osteogenic specific genes Runx2 and Osterix was detected by qRT-PCR.Results①The fourth generation of mesenchymal stem cells was positive for CD44 and CD29,while the expression of CD34 and CD45 was negative,which is in line with the characteristics of bone marrow mesenchymal stem cells.②After 3,6,9,12 d of osteogenic induction culture,the alkaline phosphatase activity of MSCs cells increased compared with the previous time point,and it was time-dependent,the difference was statistically significant(P<0.05).After 3,6,9,12 d of osteogenic induction of MSCs,the expression levels of Runx2 and Osterix gradually increased.③The AKP activity of miR-449a group and miR-449b group was lower than the control group,while the anti-miR-449 group increased AKP activity,the difference was statistically significant(P<0.05);miR-449a group and miR-449b group Runx2,Osterix expression was down-regulated compared with the control group,the difference was statistically significant(P<0.05).Conclusion This experiment successfully isolated and cultured bone marrow MSCs,which has the ability to differentiate into bone in vitro,and miR-449 can inhibit the differentiation of bone marrow MSCs into osteogenesis.