The Experimental Study On Several Methods To Induce The Marrow Mesenchymal Stem Cells Differentiate Into Myocardial Cells

Objectives: To Compare different ways about induce the marrow mesenchymal stem cells into myocardial-like cells in vitro.Methods: 6~8 weeks old SD rats femoral bone marrows were cultured to the second generation. The purity of the marrow mesenchymal stem cells (MSCs) which were examined by flow cytometry is 97%, then divided into five groups, group A: DMEM medium inducing group (control group); group B: 5-azacytidine inducing group; group C: myocardial cell lysate inducing group; group D: 5-azacytidine and myocardial cell lysate inducing group; group E: MSCs with cardiomyocytes indirect contact group. According to each experimental conditions, the MSCs were induced to 2 weeks or 4 weeks, then acquired cells. Immunohistochemical staining against cTnT, connexin43, CD31,α-SCA. Using real time PCR to investigate the expression of GATA4, NKX2.5,α-MHC,β-MHC.Results: Immunohistochemistry staining detection: The experimental groups were differentiated into myocardial-like cells expressing cTnT, CX-43 after 2 weeks cultivation in negative or low levels, but positive after 4 weeks. The Comparison between experimental groups and control group had statistic significance(P<0.05), but there are no differences between experimental Groups(P>0.05). There is no CD31 express in the control group and group B, yet group C, D, E have weak positive expressions. Before inducing, theα-SCA express in low levels in MSCs, after inducing, the concentration have obviously increase, but there are no differences between experimental Groups(P>0.05). The experimental results detected by real time PCR: There were high expressions of GATA4, NKX2.5,β-MHC in the experimental groups,α-MHC was negative. Form the density and brightness of the bands to analyze, the comparison between experimental groups and control group had statistic significance(P<0.05), but there were no differences between Experimental Groups(P>0.05).Conclusions:1. 5-Azacytidine could induce marrow mesenchymal stem cells differentiate into myocardial-like cells.2. The marrow mesenchymal stem cells have the character of"environment- dependent differentiation", and have the potency of induced MSCs differentiate into myocardial-like cells. Myocardial cell lysate group and MSCs with cardiomyocytes indirect contact group could induce MSCs differentiate into myocardial-like cells. The induced results were positively correlated with the inducing last time and the concentration of inductors. The induced cells were the cardiac possesses which between mature cells and cardiac progenitor cells.