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目的研究microRNA-129(mir-129)对骨髓间充质干细胞(bone marrow mesenchymal stem cells,BM-MSCs)向心肌分化过程中的促进作用及其机制。方法分离培养SD大鼠骨髓间充质干细胞,通过慢病毒转染使细胞获得过表达基因分为4组:对照组(MSCs);慢病毒空转组(Lentiviral vectors+MSCs,Lv-MSCs);mir-129单转染组(mir-129-MSCs);mir-129+糖原合成酶激酶(GSK-3β)双转染组(mir-129+GSK-3β-MSCs)。以10μmol/L浓度的5-氮杂胞苷(5-Aza)诱导MSCs向心肌细胞分化后,分别以第1 d、5 d、10 d、15 d和20 d为时间点,采用realtime-PCR检测GATA-4、NKx2.5、MEF-2C的mRNA水平,以第10 d、15 d、20 d为时间点,用蛋白质印迹法(Western blotting)检测肌钙蛋白I(cTnI)、结蛋白(Desmin)、GSK-3β以及磷酸化β-catenin与非磷酸化β-catenin的表达量。结果与对照组比较,随着研究时间点的推移,mir-129单转染组反映心肌分化的相关标记物基因和蛋白水平明显升高,GSK-3β的表达水平则显著降低,非磷酸化β-catenin/磷酸化β-catenin比值升高;当MSCs同时过表达mir-129和GSK-3β时,相关的心肌标记物基因和蛋白均低于mir-129单转染组,非磷酸化β-catenin/磷酸化β-catenin比值明显降低。结论过表达mir-129能够促进MSCs向心肌细胞分化,可能的机制是通过抑制GSK-3β的生成,从而削弱了对β-catenin的磷酸化抑制,后者游离入核开启调控干细胞下游的心肌分化途径。
Objective To investigate the promoting effect of microRNA-129 (mir-129) on cardiomyogenic differentiation of bone marrow mesenchymal stem cells (BM-MSCs) and its mechanism. METHODS: SD rat MSCs were isolated and cultured. Lentiviral vectors (MSCs), Lentiviral vectors + MSCs (Lv-MSCs), mir -129 single transfection group (mir-129-MSCs); mir-129 + GSK-3β double transfection group (mir-129 + GSK-3β-MSCs). MSCs were induced to differentiate into cardiomyocytes by 5-azacytidine (5-Aza) at a concentration of 10μmol / L for 1, 5, 10, 15 and 20 d, respectively. Realtime PCR The mRNA levels of GATA-4, NKx2.5 and MEF-2C were detected at the 10th day, the 15th day and the 20th day respectively. The levels of cTnI and desmin were measured by Western blotting Desmin, GSK-3β, phosphorylated β-catenin and non-phosphorylated β-catenin. Results Compared with the control group, the expression of GSK-3β in the mir-129 single-transfected group was significantly higher than that in the control group, and the expression of GSK-3β was not significantly increased -catenin / phospho-β-catenin ratio increased; when MSCs overexpressed mir-129 and GSK-3β at the same time, the related gene and protein of myocardial markers were lower than those of mir-129 single transfection group, catenin / phosphorylated β-catenin ratio was significantly lower. Conclusion The overexpression of mir-129 can promote the differentiation of MSCs into cardiomyocytes. The possible mechanism is that the inhibition of the phosphorylation of β-catenin can be attenuated by inhibiting the production of GSK-3β, which leads to the regulation of cardiac myocyte differentiation downstream of stem cells way.