| Mature myocardial tissue does not possess the ability to effectively regenerate after ischemic injury. As a result, the normal progression of injured myocardium involves development of fibrous, noncontractile scar tissue, leading to ventricular remodeling, which can cause aneurysm formation and end-stage heart failure. Cardiologists previously have mastered successful transitions between basic science and clinical cardiovascular medicine in disciplines as diverse as phannacology, imaging, and surgery. However, these interventions are limited because they are unable to reconstruct the injured myocardium. Transplanting reserve cells into an infarcted region of myocardium offers the hope of restoring some ventricular function. This technique has been labeled cellular cardiomyoplasty. The aim of this study is to explore an experimental method for primary culture of adult rat skeletal myoblasts and to investigate their function after being auto-transplanted.Methods:1. Primary skeletal myoblasts were isolated from Sprague-Dawley rats and cultured by tissue culture method. Plastic dishes and Ham?s F-b medium were used for skeletal myoblasts?proliferation. 2. After the primary myoblasts were obtained, they were transfected with retrovirus carried eGFP gene. Transfected cells in the medium were identified by fluoroscopy and fluorescence-activated cell sorter (FACS). Several factors such as the concentration of virus medium and the times of transfection repeated which would influence the transfection were also studied. 3. Approximately 7 X 106 skeletal myoblasts were expanded in vitro from adult Sprague-Dawley rats whose cardiac apexes were cryoinjured to create a transmural lesion. Two weeks after cryoinjury, autologous myoblasts or serum-free culture medium was injected into the scar tissue of transplantation(n=20) and control (n= 10) animals, respectively. Four weeks after cryoinjury, heart function in the transplantation and control groups was measured using a MPA-V instrument. Hearts were harvested and histologic studies were performed to quantify the transplanted cells. Also approximately 7 X 106 skeletal myoblasts were auto-transplanted in subcutaneous connective tissue of the adult Sprague-Dawley rat hindlimbs (n=6). The other hindlimbs were injected with 0.25 ml of serum-free Ham?s F-10 medium.Results:1. Ham?s F-10 medium nutrient mixture supplemented with 0.5% chicken embryo extract and 15% calf serum permited the purification and proliferation of rat skeletal myoblast from primary cultures. Using tissue culture method, skeletal myoblasts emerged from the tiny muscle tissue in two days, and in this condition the number of skeletal myoblasts from 1 cm3 muscle tissue reached 1 0~ per milliliter. In addition, when Ham?s F-10 was replaced with differentiation medium, the cells readily differentiated to form liner and branching multinucleated myotubes. Immunocytochemical analysis showed that over 90% cells were stained positive for myogenin, which is a factor specific to skeletal myocytes. The cell populations had a doubling time of 24h. 2. The eGFP gene transfected to myoblasts by retrovirus vector could be expressed validly. FACS analysis demonstrated that about 12.82 % adult rat skeletal myoblasts could be infected after cocultivation with producing cells. The concentration of virus medium was a major factor concerning transfection efficiency and repeated transfection could enhance the production of recombinant protein. 3. Myoblast transfer did not incur further morbidity. After cryolesion, grossly, a 0.5cm epicardial hemorrhagic lesion could be seen. Histologically, the transmural lesion contained inflammatory cells and active scarring but no viable cardiomyocytes. Within the cryolesion, eight hearts contained myocytes, which immunohistochemical analysis confirmed their skeletal muscle origin. Multinucleated myocytes were also found in the adult rat subcutaneous connective tissue of two hindlimbs. Comparing with the control group, transplantation group had lower LVW (796 ?23 vs 837 +38,...
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