| Part oneObjective: To investigate the isolation and culture of rat bone mesenchymal stem cells(BMSCs) invitro. To select a suitable inducing concentration.2. To study cardiac differentiation of human embryonic stem cells were induced by5-aza-dC,HGF and the best inducement concentration.Methods:MSCs were isolated from rat bone marrow using anchoring culture, 5-azacytidine(5-aza) for myocardial induction was added to the culture medium, the cell morphologywas noticed, Differentiation was identified by immunochemistry technique.Results:After BMSCs were treated with 5-aza 24 hours, death cells in 10μmol/L group weresmall amount. After 10 days, the cellular mophology began to change. The direction of thecells arraying was similar gradually. Passage4(P4) MSCs showed formation of myotube.After 28 days, The cells became fewer, and stretched out many long pustute. But afterBMSCs were treated with 30μmol/L 5-aza 5 days, the cells wet all deaded.Conclusion:MSCs could be induced to differentiate into cardiomyocyte-like cells by 5-aza. Fourpassage BMSCs were treated with 10μmol/L 5-aza was the optimal method for BMSCsinduced into cardiomyocytes. MSCs induced with 5-aza form myotubes, express specialimmunohistochemical phenotypes.Part twoObjective:To study cardiac differentiation of human, embryonic stem cells were induced by 5-aza-dC,HGF and the best inducement concentration.Methods:1. hESC (PKU-1-1) were planted into low adherent 6-well plates formed embryoidbodys in suspension culture for 7 days,cells were transferred to cell culture dishes toallow attachment. During the inducing process, 5-aza and HGF were used as inducingreagent, and made up many kinds of differentiating medium which are group no-reagent,group 5-aza(2.5μmol/L,5μmol/L,10μmol/L,20μmol/L), group HGF(1ng/ml,5ng/ml,10ng/ml,30ng/ml). Observing the quantity of embryoid bodys which showed spontaneouscontracting cells.2. Differentiation was identified by immunochemistry technique,RT-PCR andtransmission electron microscopy. Student's t-test was applied as appropriate and a valueof P＜0.05 was considered significant. To investigate the possible mechanisms of inducibleaction of HGF involved in the differentiation of ES cells into cardiomyocytes. For makingsure it, the expression of cardiac developmental-dependent genes were detected byRT-PCR.Results:1.0-10μmol/L5-aza-dC could induce differentiation of human embryonic stem cells tocardiomyocyte. The expressions of Troponin T and Connexin43 at protein level werepositive in 5-aza-dC-treated hESC. At the same time clear sarconeres structure wasvisible in these differentiated cells under microscope. RT-PCR showed that these cellsexpressed MLC-2A,MLC-2V,hANP,β-Actin. electron microscopy showed sarcomericorganization. The ratio of differentiation of human embryonic stein cells tocardiomyocyte in 2.5μmol/L,5μmol/L group was not significantly,and were significantlyhigher than that of 0μmol/L,10μmol/L group. in addition, 0μmol/L,10μmol/L group wasnot significantly.2.1ng/ml,5ng/ml,10ng/ml,30ng/ml HGF could induce differentiation of humanembryonic stem cells to cardiomyocyte. The expressions of Troponin T and Connexin43at protein level were positive in 5-aza-dC-treated hESC. At the same time clearsarconeres structure was visible in these differentiated cells under microscope. RT-PCRshowed that these cells expressed MLC-2A,MLC-2V,NKx2.5,GATA-4,a-MHC,β-Actin.. electron microscopy showed sarcomeric organization. The ratio ofdifferentiation of human embryonic stein cells to cardiomyocyte in 5ng/ml,10ng/ml group was not significantly, and were significantly higher than that of 1ng/ml,30ng/mlgroup.Conclusion:Human embryonic stem cells could be induced to differentiate into cardiomyocyte-likecells by 5-aza and HGF. hESC were treated with 2.5μmol/L-5μmol/L 5-aza was theoptimal method for hESC induced into cardiomyocytes,and the optimal concentration ofHGF was 5ng/ml-10ng/ml.
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