| Background and Aims Various etiologies can cause inreversible injury to cardiac cells, which were replaced by fibroblasts to form scar tissue successively because cardiomyocytes lack the ability of regeneration after birth. Quantitative deficiency of cardiac musles and progressive ventricular remodeling led to chronic heart failure. How to compensate the myocardium loss is a big obstacle that should be strode over by cardiologists. Transplantation of cultured cardiomyocytes into damaged myocardium has been proposed as a future method of treating heart failure. Mensenchymal stem cells have multi-differentiate potential ability, so they give us a new field and a new method in myocardium regneration. There are only a few number of mesenchymal stem cells in bone marrow. Acquirement of plenty of mesenchymal stem cells is a prerequisite for clinic application. In 1999, Makino et al first induced MSCs to differentiate into myocardiocytes with 3μmol/ L of 5-azacytidine treatment for 24 hour. Though it was the first time to differentiate mesenchymal stem cells into cardiomyocytes in vitro successfully, there are still many problems unsolved, such as the difficulty to reduplicate induction, low ratio of differentiation and immature condition for induction. Some study reported that electromagnetic fields not only promoted the ability of proliferation, but also accelerated differentiation of embryonic stem cells into cardiomyocytes. But whether it has influence on the differentiation of rat mesenchymal stem cell into cardiomyocytes is still unclear. Our experiments aims to optimize the condition for isolation, purification, culture of rat mesenchymal stem cells, and to observe the cell biological specificity, explore the optimal condition for the differentiation of rat bone marrow mesenchymal stem cells into cardiomyocyte cells in vitro, and to investigate the effects of pulsed electromagnetic fields on proliferation and differentiation of cardiomyocytes in rat marrow mesenchymal stem cells. Method 1. To investigate the effect of rat age on the ratio of clone forming unit and proliferation capacity of rat marrow mesenchymal stem cells through comparison with different rat age and purification methods. To explore the optimal condition for the differentiation, isolation and culture of rat bone marrow mesenchymal stem cells in vitro, and to identify bone marrow mesenchymal stem cells by examination of the cell surface antigens CD44 and CD105 with immunochemical technique. 2. The MSCs were purified by percoll mediated density gradient centrifugation, and then the third generation MSCs were randomly divided into A (with 5μmol/L 5-azacytidine treatment), B (with 10μmol/L 5-azacytidine treatment) and C (with 20μmol/L 5-azacytidine treatment) groups. MSCs in each group were treated with 5-azacytidine for 12, 24 and 48 hours, and cultured for 28 days before examination. The morphological changes in MSCs were observed with inverted microscopy. The expression of α-actin and cTnT were determined by immunohistochemistry. And the protein expression of cTnT was determined by Western blotting. 3. The third generation MSCs induced by 5-azacytidine for 7 days were exposed or unexposed to 50Hz pulsed electromagnetic fields,and then were randomly divided into A (under 0.5mT inductance intensity), B (under 1mT inductance intensity) and C (under 5mT inductance intensity) groups. These groups were divided into subgroups 1 (exposed for 10min), 2 (exposed for 20min), 3 (exposed for 30min), 4 (exposed for 10min). Four days after exposure to the pulsed electromagnetic fields, the proliferation capacity of rat marrow mesenchymal stem cells was examined by MTT method. Cell cycle was detected by flow cytometry after 14 days exposure to pulsed electromagnetic fields. And the protein expression of cTnT was determined by Western blotting so as to study the effects of pulsed electromagnetic fields on the differentiation of cardiomyocytes in rat marrow mesenchymal stem cells. Results 1. The ratio of clony forming unit and proliferation capacity in rat of two months were significantly higher than those of six months. Compared with percoll mediated density gradient centrifugation technique, marrow adhesion screening method provided considerable high clony forming efficiency, while the cell component was various. With the growth of MSCs, the cell component became simplified in the third passage, and in thispassage, almost all cells could be detected expression of CD44 and CD105 positively. 2. Twenty-eight days after induction with 5-azacytidine, scattered myogenic structure was observed in the plasma of MSCs, and positive expression of α-actin and cTnT could be detected using immunohistochemistry method. The expressions of cardiac troponin T in 5μmol/L (48h) and 10μmol/L (24h) groups was as high as those of 10μmol/L (48h) and 20μmol/L (12,24 and 48 h) groups, meanwhile the MSCs was negligibly affected. 3. After 4 days exposure to 50Hz pulsed electromagnetic fields, MSCs induced by 5-azacytidine under 0.5mT and 1mT inductance intensity for 20min～30min grew rapidly versus unexposed control groups, while growth rate was restrained significantly under 0.5mT and 1mT inductance intensity for 60min versus control groups. After 14 days exposure ,cell cycle S+G2%was decreased under three kinds inductance intensity for 60min and 5mT over 30min,but increased under 0.5mT and 1mT inductance intensity for 20min～30min. 4. The expressions of cardiac troponin T elevated significantly under 1mT and 5mT inductance intensity for 20min～30min and reduced significantly under 5mT inductance intensity for 10min. Conclusions 1. MSCs could be induced to differentiate into cardiomyocyte-like cells by 5-azacytidine in vitro. Treated with 5μmol/L of 5-azacytidine for 48 hours and 10mol/L of 5-azacytidine for 24 hours are the optimal inducing condition for MSCs differentiation in vitro. 2. Fifty Hz pulsed electromagnetic fields could promote MSCs proliferation, MSCs induced by 5-azacytidine under 0.5mT and 1mT inductance intensity for 20min～30min proliferated rapidly versus unexposed control groups 3. Fifty Hz pulsed electromagnetic fields could promote MSCs to differentiate into cardiomyocytes, and 0.5mT ,1mT and 5mT inductance intensity for 20min～30min exerted promoting effect obviously.
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