| BackgroudIschemic heart disease is one of major cause of mortality and morbidity worldwide. The symptoms of ischemic heart disease are angina, myocardial infarction, arrhythmia and heart failure, which are the consequence of decreased patency of atherosclerotic vessels. The mortality of heart failure can reach 60% within one year for patients in New York Heart Association functional class IV. Over the past decades, improvements in medical therapy, percutaneous transluminal coronary angioplasty(PTCA), coronary artery bypass surgery(CABG) and heart transplantation, have dramatically improved the prognosis of ischemic heart disease. However, the well-known limitations of these approaches , among which are complications of medicine, inconsistent efficacy of CABG and PTCA when the scar is akinetic rather than dyskinetic, high rate of severe complications of permanent implantable assist devices ,organ shortage and complications of immunosuppression in the case of heart transplantation, justify the search for alternate therapeutic options.Under this condition, someone puts forward the notion of cellular cardiomyoplasty (CMM) or cell therapy. CMM, in which one delivers appropriate donor cells into the injured myocardium, targets the basic pathophysiology of ischemic myocardium and represents a novel means of augmenting the myocyte number and contractile function.Fetal cardiomyocytes, fetal stem cells, skeletal muscle cells which were known as satellite cells, and bone marrow mesenchymal stem cells(MSCs) have been transplanted into the animal myocardium. MSCs can be obtained from bone marrow repeatedly by bone marrow aspiration and proliferate rapidly in vitro. MSCs also possess multipotential differentiation and are capable of differentiating into both mesenchymal and nonmesenchymal lineages. If enough number autologous MSCs were used as donor cells in CMM, it is not necessary for immunosuppression medicine and there is no ethic problem after transplantion. Because of these characters, the clinical use of MSCs for CMM appears to be much more advantageous.In this study, we isolate, culture and identify MSCs in vitro and explore their biolgical properties. To determine the potential to differentiate down the myogenic pathway, we provide evidence that MSCs, treated with 5-azacytidine, can differentiate into myocytes in vitro. The former studies can establish the basement of the clinical use of MSCs. To investigate the possibility of the clinical use of autologous MSCs, we infuse autologous MSCs into animal ischemic myocardium and demonstrate that implanted MSCs can improve cardiac function and express cadiomyocyogenic phenotypes under milieu-dependent differentiation. Methods:1, We isolated MSCs from animal bone marrow and separated MSCs by Percoll Centrifugation. MSCs were cultured and subcultured in low-glucose Dulbecco's modified Eagle's medium(DMEM) with 10% selected fetal calf serum. Cell growth patterns were evaluated by MTT test. Cell cycle and surface antigenic features were analyzed by flow cytometry technique.2, MSCs were treated with 5-azacytidine. We tested treated MSCs by reverse transcriptase-PCR(RT-PCR), Western Blot analysis, transmission electron microscope and immunofluorescence staining technique.3, One week after coronary artery occlusion, autologous labeled with bromodeoxyuridine(BrdU) MSCs were implanted into myocardial infarct site via topical injection. To identify and test differentiated MSCs in myocardium, we used Laser Scanning Confocal Microscopy to detect MSCs by double immunofluorescencestaining technique on myocardium histological sections 4 weeks later after transplantation. To evaluate cardiac function, we made echocardiogram examination on animal. In addition to, a micro-tip pressure transducer catheter was inserted successfully into the left ventricle chamber. Left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP) and LV peak velocities of contraction and relaxation (±dp/dtmax) were measured. Results:1 , To isolate MSCs from bone marrow aspiration, the...
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