Primary Research For Molecular Regulative Mechanism In The Process Of MSCs Differentiating Into Cardiomyocytes In Vitro And In Vivo

Objective: To investigate the temporal expression of regulative genes in the process of bone marrow mesenchymal stem cells (MSCs) differentiating into cardiomyocytes in vitro and in vivo, screen some important genes in the differentiation and reveal the molecular regulative mechanism in the differentiation.Methods: MSCs were isolated from bilateral thighbones and tibias of Wistar rats, purified by adhesive-screening method, and expanded as indifferentiated cells in culture for more than 8 passages. In vitro, MSCs were treated with 10(mol/L 5-azacytidine for 24 hours and were maintained for eight weeks. In vivo, MSCs were directly injected into the normal myocardium. Induced MSCs and myocardium specimens were obtained at 0 day, 1 day, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, and 8 weeks respectively after 5-aza treatment or transplantation. The differentiation of MSCs was observed by phase-contrast microscope, fluorescence immunohistochemistry and semi-quantitative RT-PCR from one week to eight weeks.Results:1) Purified MSCs were acquired by adhesive-screening method. MSCs were kept rapidly expanded and undifferentiated. Immortalized cells were obtained by frequent subculture for more than 8 passages. 2) Under the phase-contrast microscope, MSCs showed a fibroblast-like morphology, but the morphology changed after induction. They formed a ball-like or stick-like morphology at 1 week, connected with adjoining cells, aligned in a striated pattern after 2 weeks and formed myotube-like structure at 3 weeks. MHC began to be expressed at 1 week after induction and significant from 2 weeks after induction. The rate of positive cells is 30% at 8 weeks after induction. Connecxin43 was expressed weakly before induction and significantly after induction. The rate of positive cells is 40% at 8 weeks after induction. The expression level of TGF-(, Nkx-2.5, GATA-4 and MEF-2C genes mRNA was low before induction, began to increase at 1 day after induction, reach the peak at 1 weeks and kept high expression level after 1 week. The expression level of TEF-1 and RAR( genes mRNA kept invariant. 3) With HE stained, the MSCs gradually differentiated into cardiomyocytes from small, round, and undifferentiated cells in close approximation to intact host cardiomyocytes after grafted into normal myocardium. MHC-positive cells were detected at 1 week and connexin43 were detected between the host cardiomyocytes and transplanted MSCs at 4 weeks. The expression level of Nkx-2.5 and GATA-4 genes mRNA began to increase at 1 day after treatment, reach the peak at 1 weeks and kept high expression level after 1 week. The expression level of TGF-β,MEF-2C,TEF-1 and RAR( genes mRNA kept invariant.Conclusion: Nkx2.5 and GATA4 genes probably play important roles in the process of MSCs transdifferentiating into cardiomyocytes in vitro and in vivo; TGF-( and MEF-2C genes probably play important roles in vitro.