| Objective:To observe the primary and the passage culture process of bone marrow-derived mesenchymal stem cells (BM-MSCs); To erect the myocardial infarction model of Wistar rats; To detect the survival of Y chromosome labeled bone marrow-derived mesenchymal stem cells of male rats in ischemic myocardium of female rats; To investigate the effects of mesenchymal stem cells transplantation through epicardium and via caudal vein on the function of heart with myocardial infarction model.Methods:(1) MSCs from male Wistar rats were isolated and cultured in vitro by using the whole bone marrow adherence method. The third generation of MSCs, as the transplanting cells were obtained through isolation, purification and amplification.(2) Erecting myocardial infarction (MI) model of Wistar rats:Myocardial infarction was induced by occluding the proximal left anterior descending artery (LAD) in 100 female rats,59 of whom were the suceessful models of MI after the detecting of the echocardiogram.(3) The models of MI grouping and the cell transplantation:Two weeks after MI, the model rats were then randomly divided into 3 groups. The epicardiac injection of MSCs group (group A,n=20); the MSCs transplanting through caudal vein group (group B,n=20); the control group (group C,n=19). The MI rats in group A were opened again. The MSCs were injected into the border zones of infarcted myocardium. The MSCs were transfused into MI rats via caudal vein in group B while equivalent culture medium was transfused into group C.(4) Detecting the survival bone marrow-derived mesenchymal stem cells of male rats in ischemic perimyocardium. PCR was used to detect the sry gene which is a specific marker of Y chromosome of male rats in ischemic myocardium; Real-time PCR was used to detect the numbers of sry genes, the same as of survival MSCs of male rats in 1day,1 week,2 weeks and 3 weeks after MSCs transplantation in group A and group B.(5) Echocardiogram detect the heart function in group A,B and C before transplantation, in 2 weeks and 4 weeks after MSCs transplantation. The main parameter include left ventricle end-diastolic diameter (LVEDD), left ventricle end-systolic diameter (LVESD), left ventricle ejection fraction (EF), left ventricle fractional shorting (FS).Results:(1) The isolation, purification and identification of MSCs:The primary culture MSCs were mainly fusiform in shape. The MSCs over subcultures kept a high potential of growth and amplification. At the third passage, MSCs surface markers were detected using flow cytometry. MSCs were positive for CD 44 (92.1%±2.5%) and CD 90 (94.6%±2.1), but negetive for CD 45 and CD 34.(2) The survival bone marrow-derived mesenchymal stem cells of male rats can be detected in ischemic perimyocardium of famale rats. The specific marker of Y chromosome of male rats (the sry gene) can be detected using PCR in both group A (intramyocardiac injection of MSCs group) and group B (the MSCs transplanting through caudal vein group). Both group A and group B were positive for the agarose gelmapping of PCR product 1 week after transplantation. While no sry gene were detected in group C (the control group).(3) Real-time PCR was used to detect the numbers of sry genes in ischemic myocardium of famale MI rats. The numbers of survival MSCs of male rats in lday,1 week,2 weeks and 3 weeks after MSCs transplantation in group A were 4806±1943, 734±213,163±43,147±33, respectively. The number in group B were 77±21, 169±25,149±46,129±44, respectively.(4) Echocardiogram detect the heart function:The left heart functions in group A, B and C were obviously reduced compared with the healthy rats. Echocardiogram detected the expanding of left ventricle, the thinner anterior and less movement. The parameter of LVEDD and LVESD become bigger and LVEF and LVFS become lower. 2 and 4 weeks after transplantation the left heart functions got worse in group C while left heart functions in group A and group B were more obviously improved compared with those of before transplantation.Conclusion:(1) Both intravenous and via epicardium transplantation of MSCs can migrate into the ischemic myocardium.(2) Real-time PCR is a useful method to detect the survival number of the transplanted MSCs. There was steady growth of MSCs round the infarcted area both intravenous and via epicardium transplantation, which have protection on the rats heart after MI.(3) The effect of treatment on the heart function in MI rats was remarkable. The parameter of the left ventricular function was obviously improved compared with those of before transplantation. |
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