| Cardiovascular diseases, with increasing incidence and many unknown mechanisms, have becoming a major risk to human being. The congestive heart failure (CHF) is the last common syndrome of these cardiovascular diseases, which result in reducing quality of life and even increasing rate of death. There are several repairing mechanisms that have been described in human heart, but they cannot prevent damage to the heart from many kinds of diseases in different acute or chronic pathological conditions. Moreover, there is not a dramatic therapy to treat it; CHF remains a serious problem in clinic. Since there is growing evidence that bone marrow stem cells, such as mesenchymal stem cell, can generate new cardiocytes in the heart of animal and human, transplanting bone marrow stem cell (MSCs) to the damaged heart may provide new means to repair myocardium and prevent CHF after infarction and other diseases. The purpose of this study is to investigate the efficacy and mechanisms of transplanting autologous MSCs to canine heart with acute myocardial infraction. The study was divided into four parts as bellow.Part I : Biological and Cellular Features of Mesenchymal Stem Cells fromCanine Bone MarrowObjective: To establish the method for isolation, culture, proliferation and identification of canine SMCs from dogs. Methods: 10ml canine bone marrow aspirates, taken from the iliac crest of health canines, were diluted 1:1 with Dullbecco's Modified Eagle Medium (DMEM) and centrifuged at 900g for 10min at room temperature. The washed cells were re-suspended in DMEM to a final volume of 10 ml and layered over an equal volume of 1.063 g/ml Percoll gradient solution. After centrifugation at 600g for 30 minutes, the mononuclear cells (MNCs) were recovered from the gradient interface and washed with PBS. Percoll fractionated MNCs or nonfractionated bone marrow cells were suspended in DMEM containing lg of glucose supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 ug/ml streptomycin, and 25 fig/ml amphotericin B. All cells were plated in 10ml of medium in a culture dish. The cultures were maintained at 37C in 5% CO2 in air, with an initial medium change at 24 hours after initialplating and then medium changes every 3 or 4 days. The MSCs were identified by special cellular surface antigens and pluripotent committing differentiation potential. Results: The mononuclear cells from canine bone marrow were separated on Percoll gradient sedimentation solution at 600g for 30 minutes in small percentage (about 0.01-0.001%). The MSCs were set very good in culture of LG-DMEM supplemented with 10% selected fetal bovine serum. MSCs attached and grew as fibroblastic cells at 24 hours after initial plating, about 10"6 out of mononuclear cells. The hematopoietic stem cells that did not attach to the dish were washed from the culture with each medium change. MSCs grew rapidly, developed into visible symmetric colonies at about 3 days and reached confluence at 7 to 10 days. While the cells were permitted to proliferate to confluence, MSCs were spindle-shaped morphology, small and arranged like pectinate. As many as 1.1-1.5 X 107 cells were generated by passage 3 from a 10ml marrow aspirate. The MSCs did not differentiate spontaneously during culture expansion and maintained the normal mesenchymal stem cells biological features. These expanded attached MSCs were positive for SH2, and negative for CD45 surface antigen. The MSCs have shown the ability to give rise to a variety of differentiated cell types such as cardiomyocytes, adipocytes and osteocytes. MSCs were induced to differentiate to myotubes and myocyte-like cells by means of culture-incubated with 10~20u.mol/L 5-azacytidine. Myocyte antigens such as a-actin, oc-actinin, myosin, and cardiomyocyte specific troponin I of differentiated cells were stained positively in immunohistochemistry. Transmission electron microscope showed that differentiated cells with oval nuclei positioned in the central part of the cell had well-organized sarcomeres and myofilaments. Conclusions:...
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