LncRNA-Braveheart promotes the differentiation of bone marrow mesenchymal stem cells in vitro into cardiomyocyte-like cells / 中国组织工程研究

ABSTRACT

BACKGROUND:Our previous work demonstrated that bone marrow mesenchymal stem cells (BMSCs) transplantation could improve cardiac function in rats with myocardial infarction.However,the overall efficacy was unsatisfactory,and there was a low efficiency of BMSCs differentiating into cardiomyocytes in the local infarct myocardium.OBJECTIVE:To transfect long non-coding RNA-Braveheart (IncRNA-Bvht) into BMSCs in order to observe whether it could promote cardiomyocyte differentiation of BMSCs in vitro.METHODS:pLVX-IRES-ZsGreen1-IncRNA-Bvht vector was constructed and applied to transfect IncRNA-Bvht into bMSCs,and then,the transfection efficiency was detected.BMSCs were obtained from C57BL/6 mice and cultured ir vitro.Passage 3 cells were divided into three groupsBMSCs group,null vector group and IncRNA-Bvht group.All cells in the three groups were cultured in the normal condition for 48 hours and cardiomyocytes differentiation was induced by 5-azacytidine for another 24 hours followed by 2-week culture under normal conditions.Cardiomyocyte differentiation of BMSCs was observed under fluorescence microscopy and expression of cardiac specific cell markers including troponin T and myosin were examined using immunofluorescent staining,western blot assay,and qRT-PCR.RESULTS AND CONCLUSION:Cell morphological changes could be observed in all the groups 2 weeks after the induction.Interconnected cells arranged consistently in all the three groups.Immunofiuorescent staining results showed that the expression of troponin T and myosin was notably positive,and the proportion of troponin T positive cells was significantly increased.qRT-PCR and western blot assay results indicated that there were significantly increased levels of troponin T and myosin in the IncRNA-Bvht group as compared with the BMSCs and null vector groups (P ＜ 0.01),suggesting that IncRNA-Bvht could efficiently promote cardiomyocyte differentiation of BMSCs in vitro.