An Experimental Study On Rat Bone Marrow Mesenchymal Stem Cells Induced Committedly Differentiating Into Cardiomyocytes

That scar formation and cardiomyocytes loss result from myocardial infarction (MI) is an important cause affecting heart function, and is also the pathobiological basis leading to heart failure and even death. Recent studies show: although some cardiomyocytes begin mitotic division after MI, the number is minute and the damaged myocardium cannot be repaired completely. Today all kind of methods to treat MI such as drug, interventional and surgical therapy only can better myocardium ischemia but cannot rescue necrotized myocardium. Cardiac transplantation can replace the damaged heart, but the difficult chosen of donor, the complex technics and the high cost prevent its widely use. Effective gene therapy also depends on certain number of functional cardiomyocytes. Among bone marrow stromal cells, bone marrow mesenchymal stem cells (BMMSCs) are a group of cells with highly capability of self-renewal and potential of multilineage differentiation. They adhere to culture plates in vitro and can differentiate into mesoderm original tissues such as bone, cartilage, tendon, muscle, fat and etc, as well as ectoderm original cells such as neuron and astrocytes under certain conditions, which implies their more extensive potential of differentiation. The specific marker of BMMSCs has not been screened up to now, so BMMSCs are identified by the appearance of differentiation phenotypes in culture. BMMSCs are easy to be got and cultured in vitro and suitable for self-transplantation, which makes them an appropriate source of cells for cell therapy. To investigate BMMSCs committedly differentiating into cardiomyocytes has great theorial and clinical significance for cell transplantation replacingnecrotized and/or apoptosized ones to treat MI. Identifying sarcomeric actin and connexin43 by immunocytochemical staining is an important method to evaluate rat BMMSCs committedly differentiating into cardiomyocytes. Sarcomeric actin is a kind of contractile protein expressing in myocardium and skeleton muscle, while connexin43 is a portion of gap junction and now is a general target identifying intercalate disc. How BMMSCs are induced to differentiate into cardiomyocytes, the suitable dose of inducer, and the growth character of formed cardiomyocytes have not been demonstrated completely. Some primary researches have done abroad, but there are few related data in China.The purposes of this study: l.Set up the mode of rat cardiomyocytes differentiation by inducing BMMSCs to differentiate committedly in vitro; 2.Determine the condition of BMMSCs differentiating into cardiomyocytes by identifying myocardium marker: sarcomeric actin and connexin43 by immunocytochemical staining; 3.Explore suitable dose of inducer; 4.Draw the growth curve and division index curve of cardiomyocyte-like cells to understand the growth character of these cells and provide experimental basis for clinical cell transplantation. Materials and methods:1. Isolation and culture of rat bone marrow stromal cells: Isolate bone marrow stromal cells by their character of adherence to culture plates. 2.5% pentobarbital 40mg/Kg was given intraperitoneally to anesthesize one-month old female SD rat. Extract bone marrow specimens in sterilized environment. Cells were centrifuged at 1500rpm for 10 minutes to drop upper clear liquid and then were washed twice or 3 times with PBS and seeded at a density of 2 X 106 nucleated cells/ml to DMEM-LG medium containing 10% fetal bovine serum (Falcon 3002 dishes diameter 60mm area 21cm2) and cultured at 37癈 in humidified atmophere of 5% CO₂. 24 hours after seeding, medium was changed for the first time, and afterwards medium was changed every 3-4 day. When the attached cells grew to full of plates' bottom, the cells were released from the dishes for 3-4 minutes at room temperature with 0.25% trypsin and centrifuged at l000rpm for 5 minutes. The cell pellet was then resuspended at a density of 1X 105cells/ml to passage.2.Ascertain the suitable dose of inducer: 48 hours after second passage, cells were exposed to 5-aza.