Repair Of Infarcted Myocardium By Autologous Bone Marrow Stromal Cells Transplantation In Rabbits

Background and objectivesMyocardial infarction leads to loss of tissue and impairment of cardiac function. The remaining myocytes are unable to reconstitute the necrotic tissue, and the post-infarcted heart deteriorates with time. Although cardiac transplantation is an ideal treatment for end-stage heart disease, inadequate donor availability has stimulated efforts to manage terminally injured myocardium by other innovative methods. Autologous cells transplantation for the treatment of damaged myocardium after myocardial infarction is becoming an increasingly promising strategy. Marrow stromal cells(MSCs) retain the capacity for unlimited, undifferentiated proliferation and are capable of developing into different types of cells, including cardiomyocytes. MSCs, by contrast with other types of cells, can be collected from adults and used for transplantation without posing ethical questions or creating problems of tissue matching and rejection. Implantation of autologous MSCs in the heart might be a potential method to restore tissue viability after myocardial infarction.In this study, we tested the hypothesis that intramyocardiac implantation of MSCs may regenerate infarcted myocardium and reduce cardiac dysfunction. Moreover, we tried to confirm some factors, which had influence on the transplantation. In addition, several experiments in vitro aimed at the potential of MSCs to be transplanted in the infarcted heart.Methods and resultsIn vitro MSCs were isolated by gradient centrifugation and expanded in vitro. Flow cytometry analysis of the cultured MSCs indicated more than 95% CD34-(Hematopoetic) and CD44+(Mesenchymal). 5-bromo-2-deoxyuridine(BrdU) were added into cultured MSCs for 24 hours and more than 90% MSCs were effectively labeled with BrdU. Then MSCs were induced by 5-azacytidine with different concentration for 24 hours. After cultured for another 3 weeks, some of the induced MSCs were identified to have differentiated towards myocytes by the means of transmission electron microscope technique, immunocytochemistry technique and western blot technique. We also cocultured MSCs with direct cell-cell interaction of rats' cardiomyocytes after labeling them with BrdU. After cocultured for 3 weeks, most BrdU-labeled MSCs exhibited immunocytochemical a -actin positive expression. Degenerated cardiomyocytes and differentiated MSCs were found in this cocultural system under electron microscope. However, MSCs in another cocultural system, where MSCs and cardiomyocytes were layer-cultured, didn't show any tendency towards myocytes. Recombined DNA pcDNA3.1-VEGF165 were transfected into MSCs with the help of lipofectamine. Immunocytochemical analysis indicated that the expression of VEGF165 in these cells after transfection increased obviously.In vivo In our study, 34 New Zealand White rabbits underwent left thoracotomy for coronary artery ligation, hindlimb bone marrow aspiration and intramyocardiac injection. The rabbits were randomly divided into 3 groups according to the means dealing with MSCs and the components injected into myocardium. Group A didn't have their MSCs induced in vitro. Group B had their MSCs induced by 5-aza. Group C had their MSCs only used in experiments in vitro. Both group A and group B were injected with autologous MSCs, while group C was injected with physiological saline served as controls. Group A and group B were divided into two subgroups respectively according to the location where the cells were injected. Myocardial infarction was created in rabbits, while MSCs were isolated and expanded subsequently. Before transplantation, MSCs were incubated with BrdU for 24 hours. Then cells or physiological saline were injected into the infarcted hearts. Before and after these procedure left ventricular(LV) function were determined by echocardiography. 1 month later, histological examination under light microscope and transmission electron microscope were conducted to observe the influence of the injected MSCs on the infarcted heart. The difference in LV function between cells transpla...