| Diseases of cardiovascular system, particularly myocardial infarction, remain a leading cause of morbidity and mortality in human. Because of the increased age of the population and improved postinfarction survival rates, congestive heart failure (CHF) is the only cardiovascular disorder increasing in prevalence and incidence. Contemporary medical therapy is simply palliative and accounts for a persistently high mortality that can reach 60% within 1 year for patients in New York Heart Association functional class IV. Although cardiac transplantation remains the only radical treatment of heart failure, organ shortage and immunerejection largely limit this approach. Spontaneous terminally differentiated cardiomyocytes have shown some evidence of mitotic division in the adult heart, but the rate of proliferation is minute and cannot meet the demand for tissue regeneration. So the most realistic approach consists of exogenously supplying a new pool of contractile cells and to engraft them into the postinfarct scars --Cellular cardiomyoplasty (CCM) .CCM aims to replenish myocytes number, being based on two major assumptions: (1) CHF develops when a critical number of cardiomyocytes have been irreversibly lost, and (2) function can thus be improved by repopulating these areas of "dead" myocardium with a new pool of contractile cells. In this setting, bone marrowmesenchymal stem cells (BMSCs) with the possibility of being used as autografts and no immune-suppressive therapy necessary, are currently generating a great deal of interest. In our previous studies, BMSCs, by morphologic changes and immunocytochemical staining such as sarcomeric actin and connexin-43, had been successfully induced to cardiomyocytes in vitro with induction of 5-azacytide. However, whether BMSCs, which were treated with 5-azacydine before transplantation, could survive in the scars, differentiate into cardiomyocytes and improve ventricular dysfunction, has not been demonstrated completely. Some related studies have been done abroad, but there were few in China.Therefore, the purpose of the present study was as follows: (1) the feasibility of culture and expansion of BMSCs in vitro. (2) to investigate wether transplanted BMSCs could survive in the infracted myocardium. (3) to study whether in-scar transplanted BMSCs could transdifferentiate into cardiomyocytes in the appropriate "niche" and express cardiac specific marker sarcomeric actin. (4) to assess the functional improvement in rat left ventricular with coronary ligation afeter transplantation. Materials and methods:1. Reparation of rat BMSCs: 120- 140g SD rats, under general anesthesia, the femoral and tibial bone marrow specimens were extracted by sterile syringe containing 10% fetal bovine serum and DMEM-LG. Cell suspensions were centrifuged at 1000rpm for 10 min. Then discarded upper clear liquid and wash three times with PBS. Cells isolated from bone marrow in 5ml DMEM-LG medium, with 10% fetal bovine serum, penicillin G(100U/ml) and streptomycin(100ug/ul), were then introduced into plastic culture dishes(Falcoon 3002) and incubated with 95% air and 5.0% CO2 at 37C. The medium was completely changed 24 hours later in order to discard nonadherent cells and subsequently replaced every 3 days. Cultured BMSCs were observed under contrast-phase microscope to assess the level of expansion. When cultured cells reached about 80% confluence, the culture medium was discarded and washed three times by PBS. Then the adherent cells were released from the disheswith 0.25% trypsin and replanted to 3 dishes (first passage). After the twice-passaged cells became nearly confluent, 5-azacydine was added into the culture medium at a final concentration of 10 umol/L for 24 hours and then washed with PBS. To identify the transplanted cells in the recipient scar tissue, sterile bromodeoxyuridine (BrdU) solution was added to the culture medium at a final concentration of 0.1 mg BrdU/ml culture medium for 2 days immediately before transplantation.2. Creation of the myocardial infarction: Under g...
|
PDF Full Text Request