The Differentiation Of Rat Bone Marrow Mesenchymal Stem Cells Into Cardiomyocyte-like Cells By 5-Azacytidine In Vitro

Objective:To isolate and culture bone marrow mesenchymal stem cells（BMSCs）in vitro and the differentiation of rat bone marrow mesenchymal stem cells into cardiomyocyte-like cells by 5-azacytidine in vitro,so as to provide good seed cells for transplantation of BMSCs to treat myocardial fibrosis.Methods:（1）Bone marrow mesenchymal stem cells（BMSCs）were isolated from Wistar rat bone marrow by whole bone marrow adherent culture method and cultured in a 37℃5%CO₂ incubator.When the adherent cells approached most of the fusion,they were subcultured and amplified repeatedly.Cell growth was observed daily under inverted phase contrast microscope.The absorbance of the 2nd generation BMSCs was measured by MTT method and the growth curve was drawn.The expression of CD29 and CD45 on the surface of the 3rd generation cells was measured by flow cytometry.（2）The 3rd generation of bone marrow mesenchymal stem cells in good growth condition were induced by 5-azacytidine（control group,5 umol/l,10 umol/l,15 umol/l）for 24 hours and then cultured in 37℃5%CO₂ incubator.The morphological changes of cells were observed under inverted microscope.The expression of troponin T（cTnT）,connexin-43（CX-43）andɑ-smooth muscle actin（ɑ-SMA）were detected by immunocyto chemical staining on the 28th day.Results 1.Under inverted microscopy,the primary cells were polygonal and showed a trend of colony growth,reaching 80%-90%in about 7-10 days.The passaged cells extended in a long spindle shape,arranged in a directional direction,and grew in a whirlpool shape.They were passaged once every 4-5 days.2.The growth curve of the second generation bone marrow mesenchymal stem cells（BMSCs）was drawn by MTT method.It was found that the proliferation rate of BMSCs was the fastest on the 3rd to 5th day,and began to grow slowly or even declined on the 6th day,so the best passage time was the 5th day.3.Flow cytometry showed that the positive rate of CD29 and CD45 on the surface of the third generation bone marrow mesenchymal stem cells was 99.4%and 1.4%,indicating that the third generation bone marrow mesenchymal stem cells used in the experiment were purified cells.4.After inverted microscope and HE staining,the cells in the control group were fibroblast-like and arranged polarly.At the beginning of 7 days,some cells in the experimental group became larger and different in shape,showing irregular shape,triangle,long spindle,etc.The polarity of cells disappeared and arranged irregularly.Some cells protruded large protrusions and fused with each other,but no obvious spontaneous pulsation was observed in the experimental group within 28 days.5.On the 28th day,different concentrations of 5-azacytidine were used to induce bone marrow mesenchymal stem cells.The expression rates of cardiac special troponin（cTnT）were 13.667±3.2145%,44.3333±4.0414%,32.3333±1.1547%（P≤0.01）,connexin-43 were 6±2%、31±4%、11±1.7320%（P≤0.01）and the expression rates of a-actin were 13±4.3589%、43±4%、26.3333±1.5275%（P≤0.01）,which were significantly higher than those of the control group.Conclusions 1.Whole bone marrow culture is a simple,rapid and efficient method for obtaining high purity bone marrow mesenchymal stem cells in vitro.2.The differentiation of rat bone marrow mesenchymal stem cells into cardiomyocyte-like cells by 5-azacytidine in vitro,and the highest positive expression rate is 10 umol/l.