ABSTRACT
BACKGROUND: Our previous study demonstrated that bone marrow mesenchymal stem cells (MSCs) presented with a low survival rate and newly formed vascular-like structures was sparsely distributed in the local infarct tissues after cell transplantation, which certainly impaired the therapeutic efficacy. Long non-coding RNA-H19 (lncRNA-H19) has been confirmed to be associated with MSCs differentiation and mediate vascularization. OBJECTIVE:To observe the influence of lncRNA-H19 on the survival and vascularization potential of MSCs in vitro and to explore the possible mechanism. METHODS:MSCs were obtained and cultured in vitro.Cells were divided into five groups:MSCs+H19,MSCs+H19 negative control (MSCs+H19 NC), MSCs+si-H19, MSCs+si-H19 negative control (MSCs+si-H19 NC) and MSCs groups. MSCs+H19 and MSCs+H19 NC groups were transfected with lncRNA-H19 and lncRNA-H19 scramble RNA respectively, while MSCs+siH19 and MSCs+si-H19 NC groups were transfected with lncRNA-H19 siRNA and lncRNA-H19 siRNA scramble respectively. Cells were cultured under hypoxic-ischemic condition (serum-free medium, 1% O2) for 24 hours. Then, cell proliferation and apoptosis were evaluated using MTS and TUNEL, respectively. Cell supernatant from each experimental group was further co-cultured with human umbilical vein endothelial cells to induce vascularization. The expression of vascular endothelial growth factor A (VEGFA) was thereafter detected using western blot assay. RESULTS AND CONCLUSION: Compared with MSCs+H19 NC and MSCs groups, MSCs+H19 group presented with significantly higher proliferation rate, lower apoptosis percentage and a larger number of vascular branches on matrigel (P < 0.01). There was a significantly higher expression of VEGFA in the MSCs+H19 group than MSCs+H19 NC and MSCs groups. Compared with the MSCs and MSCs+si-H19 NC groups, MSCs+H19 group presented with significantly lower proliferation rate, higher apoptosis percentage and a less number of vascular branches on matrigel (P < 0.01). In addition, VEGFA expression was distinctly downregulated in the MSCs+si-H19 group in comparison with the MSCs+si-H19 NC and MSCs groups. These findings indicate that lncRNA-H19 effectively promotes MSCs survival and vascularization under hypoxic-ischemic condition in vitro,and this effect may be associated with the upregulation of VEGFA.
ABSTRACT
BACKGROUND: Our previous studies demonstrated that cardiac stem cells (CSCs) transplantation could improve cardiac function in rats with myocardial infarction (MI). However, the overall survival and cardiac differentiation of CSCs were low. OBJECTIVE: To investigate the effect of hypoxia preconditioning on CSCs proliferation and cardiogenic differentiation and the role of hypoxia induced factor-1alpha (HIF-1α)/apelin/putative receptor protein related to the angiotensin receptor AT1 (APJ) pathway in the procedure. METHODS: Cells cultured in vitro experienced exposure to hypoxia (1% O2) for 24 hours. Cardiogenic differentiation was induced by using 5-azacytidine for another 24 hours. Then, cells were cultured in normal condition for 2 weeks. Normoxia (20% O2) was used as a negative control during the whole process. Cell proliferation was detected using MTS method and expressions of HIF-1α, apelin, cTnT and APJ were detected using western blot assay after 24 hours of preconditioning and 2 weeks after the induction of differentiation; the percentage of cTnT-positive cardiomyocyte-like cells was observed by immunofluorescence staining. RESULTS AND CONCLUSION: Compared with the normoxia group, the hypoxia group presented a higher proliferation rate and a higher absorbance value at 490 nm (P < 0.01); the protein expressions of HIF-1α, apelin and APJ were all enhanced after hypoxia exposure for 24 hours and 2 weeks after the induction of differentiation (P < 0.01); the percentage of cTnT-positive cells was greatly increased in the hypoxia group (P < 0.01), and the expression of cTnT was also significantly intensified (P < 0.01). To conclude, hypoxia preconditioning could promote the proliferation and cardiogenic differentiation of CSCs, and the activation of HIF-1α/Apelin/APJ pathway might be involved in this process.
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 groups:BMSCs 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.