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

With the improvement of people's living standard and changes of dietary structure in recent years, the incidence of ischemic heart disease has increased rapidly in China. It is a heart disease which can lead to myocardial necrosis or degeneration of the heart caused by an imbalance of the oxygen supply and the oxygen consumption. The mature myocardial tissue has no regeneration ability. After the death of myocardial tissue resulted from hypoxia, the proliferation of fibrous tissue and the formation of scar occur consequently, which leads to ventricular remodeling and congestive heart failure. The current treatments to the ischemic heart disease include drug therapy, coronary artery bypass grafting, interventional therapy arid laser treatment. However, all the measures designed to improve the local blood supply of heart and to enhance the function of surviving cardiac cells, have no effect on dead myocardial cells. This feature determines that all these treatments are not fundamental. In recent years, the research on stem cell has made remarkable achievements, and the technologies such as stem cell purification, amplification in vitro and induction, have become mature gradually. At the same time, the concept of cellular therapy comes into view. Cellular therapy is a new kind of therapy which can repair, replace damaged tissues and organs by transplanting functional cells, and it has become a new idea of treating ischemic heart disease. Autologous transplantation in myocardium is a hot topic in cardiovascular disease field in the past few years, as it can repair or replace the damaged heart function by transplanting multiple kinds of stem cells (embryonic stem cells, skeletal muscle satellite cells, bone marrow mesenchymal stem cells) into the body. By studying the differentiation of rat bone marrow mesenchymal stem cells into cardiomyocyte-like cells induced by 5-azacytidine,we investigate the methods of cell separation and purification in vitro, the biological characteristics of bMSCs, the appropriate concentration of inducer, and the identification approach. Finally, this experiment provides the theoretical basis for clinical application.ObjectiveTo explore the differentiation of rat bone marrow mesenchymal stem cells into cardiomyocyte cells induced by 5-azacytidine in vitro. To offer seed cells for Cell Therapy.Materials and methods1. Experimental MaterialsSD rats,4 weeks old, supplied by the Experimental Animal Center of Henan Province.2. Methods(1)The femur of SD rats was obtained under sterile conditions, and the separation and purification of rat bone marrow mesenchymal stem cells are achieved by different ability of adhibition.The rat bone marrow cells were cultured in low-glucose DMED with 10% fetal bovine serum. When the cells cover more than 80% of the culture flask bottom, the adherent stem cells were seperated and digested by using trypsin. To identify the mesenchymal stem cells, we detect the expression of cell surface antigen by morphological methods and flow cytometry.(2) MSCs of the Third-generation were induced with 5-Aza of 0,5,10,20μmol /L for 24 hours in vitro respectively, and then MSCs were cultured in low-glucose DMEM with 5% fetal bovine serum.(3) On the 25th day of cultivation after the inducement, the MSCs were re-inoculated into the 24-well plates and then cultured in low-glucose DMEM containing 5% fetal bovine serum.(4) On the 28th day of cultivation after the inducement, the expression of cardiac troponinⅠ(cTnⅠ) was identified by Immunocytochemistry.(5) Calculating the positive rate of cTnⅠ.Analysing the dates presented with mean±standard deviation by statistical software of SPSS 13.0.One-way ANOVA and LSD-t test were applied in dates analysis.The difference was statistically significant(p<0.05).Results(1)The MSCs separated by differential adhesion method has uniform cell size and vigorous ability of proliferation. According to trypan blue staining,the viability of cells is more than 96%.CD90 and CD44, the surface marker of Bone marrow mesenchymal stem cell, were tested by means of flow cytometric analysis.The results show that the positive rate was 98.7% and 98.5% respectively. CD34 and CD45, the surface marker of hematopoietic stem cells, have the negative results.(2)24h after inducement,some cells droped from the culture flask.One week after inducement, we could observe that a few cells became larger and fusiform. Two weeks after inducement, some cells appeared to be short columnar and multi-angular while the cells contacted closely. Four weeks after inducement, the size of cells had moderate difference and the endochylema was rough.The beating of induced cells was not observed.(3) The difference on differentiation between 10μmol/L group and 20μmol/L group was not statistically significant(P=0.639).The differentiation rate of both groups were higher than that of 5μmol/L group.Conclusions(1) The differential adhesion method, which was used to separate and purify the MSCs, is simply and effect. The cells obtained have a high purity.(2) The low-glucose DMEM containing 10% fetal bovine serum is suitable for the culture of rat bone marrow mesenchymal stem cells in vitro.(3)5-Aza can induce the rat MSCs despetated and purified in vitro into cardiomyocytes-like cells,while the cardiac specific troponinⅠwere detected in the induced cells.(4) According to the test, 10μmol/L of 5-azacytidine concentration is appropriate for generating a good effect in inducement.