The Experimental Research To Inducing Rat Mesenchymal Stem Cells Into Cardiomyocytes In Vitro

Objective: To investigate the method of rat mesenchymal stem cells separated and cultured in vitro and to observe the biologic characteristics;To investigate the method by which rat mesenchymal stem cells can be induced into cardiomyocytes and the method which can identified them.Method: 15 Wistar rats,male,weighting 180±20g were separated into two groups randomly.One group is 7 rats,another is 8 rats.All of the rats were anesthetized with pentobarbital(30mg/kg body weight)by anintraperitoneal injection.Then the rats were come off the thighbones . The stem and epiphysis of thigbones of 7 rats were rinsed with PBS ,then rMSCs were isolated by Percoll separating medium(1.073g/ml) and cultured in culture bottles.Another 8 rats were rinsed with DMEM of 15% FBS,100ug/ml penicillin and 100ug/ml streptomycin.Finally,the rinsed water were inoculated in culture bottles and were cultured in vitro,observing rMSCs biologic characteristics.Then rMSCs were induced by 5-Aza 5umol/L for 24 hours.We identified that some of the induced rMSCs had differentiated into cardiomyocytes by means of transmission electron microscopy technique,immunofluorescent technique.Results: rMSCS isolated by the whole bone marrow culture had higher purity .better uniform and ability of proliferation in the process of culture in vitro than those of Percoll isolated method.5umol/L 5-Aza is an optimal concentration for rMSCs to differentite into cardiomyocytes.Some of the induced rMSCs were positive expression of a-Actin,cardiac troponinl (cTnl).Electron microscopy revealedmyofilament-like structure,rich glycogen granules and anumber of mitochondria.Conclusion: 1 The method of whole bone marrow culture is a well-referenced medium for density gradient centrifugation of rMSCs;2 The optimal conditions for rMSCs to differentiated into cardiomyocytes is 5umol/L 5-Aza;3 Mscs are mutipotential stem cells and they can be induced into cardiomyocytes by 5-Aza in vitro which have the characterestics of cardie myocytes in structure.