| AIMS To investigate the effect of intramyocardial-transplantion mesenchymal stem cell(MSCs) and erythropoietin(EPO) for the treatment of myocardial infarction. We investigated whether EPO enhances the therapeutic potency of MSCs transplantation in rats with experimental myocardial infarction(MI).METHODS The experiment was performed at the animal laboratory and the central laboratory, The First Affiliated Hospital of Anhui Mdical University between December 2007 and March 2009.①animal: Sixty Sprague-Dawley(SD) rats were randomly divided into 4 groups:MI group,MSCs group,EPO group and MSCs-EPO group after MI was made by ligating the anterior descending coronary artery successfully. What to do with animals in the experiment is consistent with animal ethical standard.②MSCs culture,inducing and labeling in vitro:MSCs were isolated and purified with adherence plasticity method. The shape was observed and CD34, CD44, CD45, CD90 were detected with Flow cytometry analysis. MSCs were labeled by 5-bromodeoxyuridine (BrdU) and were to be applied.③MSCs transplantation and EPO infusion: Transplantation of MSC and/or 7-day infusion of EPO was performed immediately after coronary ligation. MSC(2×106 cells in 200ul saline solution) was injected into four sites, one within the infarct area and three in the myocardium bordering the ischemic area. EPO (5000U/kg body weight) was subcutaneously administered for 7 days, whereas control animals were injected saline solution for the same time duration.④experiment evaluation: Hemodynamics was performed four weeks afer operation. Then the rats were sacrificed. Cardiocyte apoptosis was determined by terminal deoxynucleotidyl transfease-mediated dUTP nick-end labeling(TUNEL) methods. Fluorescence microscope was used to identify the BrdU-labeled cells.Vascular density and the level of Bcl-2,Bax were evaluated by immunohistochemical technique.RESULTS①Cells cultured in vitro adhered obviously after 24 h, cells were many kinds of shape such as round,fusiform,and irregularity; after passages, the form of cells was becoming to be conformity, being fusiform. The reproductive activity of bMSCs cultured in vitro was strong, MSCs grew colony and the shape of cell colony was on"swirl".②After passages of bMSCs, the positive rate of CD34 and CD31 on bMSCs cell surface is below 5% while the positive rate of CD44 and CD90 is above 90%.③Four weeks after transplantation, some cells were observed in the MSCs group and MSCs-EPO group, which were positive for BrdU. The immunofluorescence staining demonstrated that transplanted cells can survive in the peri-infarction region.④Compared with those in MI group, hemodynamic indexes of rats in MSCs group,EPO group and MSCs-EPO group were significantly improved.Hemodynamic indexes of rats in MSCs-EPO group were the most significantly improved(P<0.05) .⑤the cell nucleus of apoptotic cells were buffy by TUNEL staining, and compared with MI group, cardiocyte apoptotic index is much lower than that of MSCs,EPO and MSCs-EPO group(P<0.05).⑥Capillary density was markedly higher in the MSC-EPO group, followed by MSC and EPO groups, when compared with control group (P<0.05).⑦Compared to other groups,Bcl-2 was significantly up-regulated and Bax down-regulated in MSCs-EPO group.CONCLUSIONS:①MSCs transplantation and EPO infusion could reduce cardiocyte apoptosis ,stimulate angiogenesis and improve cardiac function.②EPO enhances the therapeutic potency of MSCs transplantation. The mechanisms may be associated with the effect of EPO, which EPO can improve the cardiac micro-environments, facilitate the survival and differentiation of implanted MSCs in vivo and drive a pro-angiogenesis program within the MSCs.
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