Experimental Study Of Different Conditions On Bone Marrow Mesenchyma Stem Cells Differentiating Into Cardiomyocytes In Vitro

Objective:To investigate in vitro isolation and culture of bone marrow mesenchyma stem cells (MSCs) and cardiomyocytes(CM) in rats,whether the MSCs can differentiate to the myocardial cells in the presence of 5-Aza-2'-deoxycyti-dine and coculture with CM.After inducing and differentiating in different ways,we investigate the ability that MSCs differentiate into myocardial cells and the differences that MSCs express cardiac cell-specific markers of connexin(Cx)43 and troponin T (cTnT) in different time,so as to provide a theoretical basis for MSCs transplantation in the treatment of acute myocardial infarction (AMI).Methods:Bone marrow was sterilely collected from the bilateral thigh-bone of rat.MSCs were isolated,purified and amplified by Percoll method plus adherence method.Sections from cells assemble were identified by transmission electron microscope.CM was sterilely collected from neonatal rats.CM were isolated,purified by difference-speed adherence method and then were detected about connexin43 and cTnT by immunohistochemistry.After obtaining the stab- le MSCs line,the MSCs were labed with DAPI at the third passage.Dividing into three groups:①normal control group:DAPI-MSCs grown in ordinary medium.②5-Aza-induced group:The DAPI-MSCs were treated with different concen-tration of 5-Aza and exposed to 5-Aza for different time to observe the optimal concentration and time and then were detected about connexin43 and cTnT by immunohistochemistry.③coculture group: DAPI-MSCs were cocultured with cardiomyocytes that have been cultured for 3 days.On the 1,2,3 and 4 weeks, DAPI-MSCs were observed the cell morphology with an optical inverted microscope,identified cTnT,Cx43 by immunohistochemistry and calculated the positive rate of induction.Results:In vitro the MSCs isolated from Wistar rats had favourable reproductive activity and kept the Characteristics of undifferentiation.CM were able to spontaneous pulse after separated and adherented for 1 day.After cultured for 2～3 day,the CM could grow into a monolayer and formed cell clusters,and formed synchronization pulse of clustered cells.At the 4 week,we can observe the typical changes in the MSCs persistly exposed to 10μmol/L 5-Aza for 15days and some could show spontaneous beating with the frequency of 15～20 per min.MSCs were labed with DAPI at the third generation.The marking efficiency was 100%.DAPI-MSCs wasn't found cell shrinkage and the expression of cTnT,Cx43 in the normal medium.In the induction group and the coculture group,the cells were found that orientating with one accord,arranging into a muscular and pulsing spontaneously at 4 week.In the induction group,the expression of cTnT,Cx43 were not observed at 1 week. The positive rate of cTnT,Cx43 was (9.98±1.67)%, (13.38±2.15)% at 2 week. Compared to 2,3 weeks, the positive rate of cTnT, Cx43 was increased at 4 week (P<0.01). In the coculture group, the expression of cTnT, Cx43 were detected at 5th day and increased with time. Compared tol,2,3 weeks, the positive rate of cTnT, Cx43 was(88.3±1.33)%, (90.38±1.87)% at 4 week (P<0.01).In the same weeks, the positive rate of cTnT, Cx43 in the coculture group was higher than the rate of induced group (P<0.01).Conclusions:In vitro MSCs had favourable reproductive activity and kept the Characteristics of undifferentiation.CM were isolated and purified with a difference-speed adherence method. The cell purity was over 95%. The MSCs could differentiate to the myocardial cells and expressed cTnT,Cx43 in the presence of 5-Aza and cocultured with CM. The ability of MSCs differentiated into myocardial cells in the coculture group was stronger than in the induced group.