The Study Of Cardiomyocyte Differentiation Ability Of Rat BMSCs In Vitro And BMSCs In Myocardial Ischemia

Background: At present myocardial infarction is one of seriously endangering human being health disease. Although, many breakthroughs in pharmacological and surgical managements, modern reperfusion strategies, the complications of myocardial infarction remain a serious problem cardiomyocytes are reduced. Bone marrow mesenchymal stem cells could provide a promising strategy against myocardial infarctions and postinfarct congestive heart failure, based on bone marrow mesenchymal stem cells can generate new cardiomyocytes. The purpose of this study was to research cardiomyocyte differentiation ability of rat BMSCs in vitro and transplant BMSCs in myocardial ischemia.PartⅠThe Study of Cardiomyocyte Differentiation Ability of Rat BMSCs in VitroObjective: To isolate and culture a highly purified population of adult rat BMSCs and to analyze their biological characteristics.Methods: BMSCs were isolated and cultured from rat bone marrow with density gradient centrifugation. Trypan blue staining and cell counting were used for the cell concentration. Flow cytometry(FCM) was used to analyze the expression of CD44,CD29,CD45,CD34. To induce differentiation, BMSCs were induced by 5-azacytidine(5-aza). Immunohistochemistry analysis was used to test the expression of MHC and cTnI.Result: The BMSCs could be passaged culture continuously. FCM analysis showed that the BMSCs were CD44, CD29 positive, and CD45, CD34 negative. The passage 3 BMSCs that were induced by 5-aza could express MHC and cTnI after 2 weeks.Conclusion: BMSCs possess cardiomyocyte differentiation potential and can be differentiated into cardiomyocyte cells by 5-azacytidine.PartⅡBMSCs as Transplantation Cells for Myocardial Ischemia TherapyObjective: To investigate BMSCs as transplantation cells to improve cardiac functions in rat MI model, and to explore the underlying mechanism in vitro.Methods: The BrdU-BMSCs were co-cultured with rhythmically beating CM or cultured in the presence of conditioned media for two week. Immunohistochemistry analysis was used to test cTnI of the BrdU-BMSCs. Rat myocardial infarction model was established by ligating the left coronary artery. Cardiac function of MI was assessed by echocardiographic-Doppler. BMSCs were injected directly into cardiac muscle around the infarct zone. After 20 days, HE dyeing was used to analyze the transplantation BMSCs in the infracted area.Results: (1) The BrdU-BMSCs which were co-cultured with rhythmically beating CM showed cTnI after 2 weeks but BrdU-BMSCs cultured with culture media without cardiomyocytes did not. (2) After the cellular implantation, the stroke volume(SV) and ejection fraction(EF) [SV=(0.466±0.039)ml,EF=(0.651±0.029)%]decreased in the A group(normal group) compared with before the cellular implantation[SV=0.703±0.099ml,EF=(0.789±0.039)%]. (3) The heart function of the B group(PBS group) [SV=(0.478±0.052)ml,EF=(0.641±0.019)%]have not distinct improvement compared with the A group(P>0.05). (4) The heart function of the C(BMSCs group) [SV=(0.634±0.027)ml,EF=(0.770±0.054)%] and D(5-aza group) [SV=(0.642±0.046)ml,EF=(0.789±0.022)%] groups have distinct improvement compared with the A group(P>0.05), but there are not evident distinction between C and D group.Conclusions: Direct cell-cell contact between BMSCs and CMs was the prerequisite for the cardiomyocyte differentiation of BMSCs. The BMSCs injected directly into cardiac muscle around the infarct zone as transplantation cells can improve the cardiac function of MI. The BMSCs induced by 5-aza also can improve the cardiac function of MI. No matter whether the BMSCs were induced or not by 5-aza, the improvement of the cardiac function might result from myocardial regeneration by engrafted cells.In summary, BMSCs have cardiomyocyte differentiation potential in Vitro. Direct cell-cell contact between BMSCs and CMs was the prerequisite for the BMSCs cardiomyocyte differentiation. No matter whether the BMSCs were induced or not by 5-aza, The BMSCs can improve the cardiac function after the cells were implanted around the infarct zone and its mechanism might result from myocardial regeneration by engrafted cells.