Dissertation > Medicine, health > Internal Medicine > Heart, blood vessels ( circulatory ) disease > Heart disease

The Experimental Research to Inducing Rat Mesenchymal Stem Cells into Cardiomyocytes in Vitro

Author CaiXiaoJun
Tutor JiXiaoPing
School Shandong University
Course Internal Medicine
Keywords Bone marrow mesenchymal stem cells Myocardial cells Whole bone marrow culture Cell differentiation 5 - azacytidine Immunofluorescence surgery
CLC R541
Type Master's thesis
Year 2005
Downloads 130
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Purpose: to explore rats since bone marrow mesenchymal stem cells in vitro separation cultivation method to observe the bone marrow charge quality stem cells of the biological characteristics; explore on cultured bone marrow mesenchymal stem cells carried out in vitro drug orientation induced to differentiate into cardiomyogenic cells and identification of cardiomyocyte-like cells. Methods: We selected 15 wistar male rats, weighing 180 ± 20g, were divided into two groups, a group of seven, culture isolation, culture Percoll separation; another group of eight, separation of whole bone marrow culture, culture. 15 rats by intraperitoneal injection of pentobarbital sodium (30 mg / kg) anesthesia, under sterile conditions, breaking away from the femur, a group of seven with PBS buffer wash femoral shaft and metaphyseal then Percoll solution (density 1.073g/ml) density gradient centrifugation, take the interface turbid layer, the cells were washed 2 times with DMEM, was inoculated into the disposable plastic culture flasks; another group 8 with DMEM culture solution flushing femoral shaft and dry epiphyseal end, seeded in culture flasks, adherent isolated mesenchymal stem cells in the bone marrow. Mixed with bone marrow mesenchymal stem cell culture system (DMEM 15% PL quality fetal bovine serum, penicillin, streptomycin 100 μg / ml; 2 , at 37 ° C with 5% CO ), amplified. Be primary cultured cell proliferation to 80%, the cells were digested with 0.25% bovine trypsin into a single cell suspension was subcultured. Add to 5 - azacytidine 5μmol / L at 24 hours the take grew well in the third generation of bone marrow mesenchymal stem cells after trypsin digestion, cultured normal culture conditions, the cells covered culture bottles (about 5-6d): 2.5 passage after passage, the same induction, training, observation of cell morphology changes and the growth and development of an inverted microscope. The induced cells were identified by immunofluorescence technique, transmission electron microscopy technology. : Whole bone marrow culture method compared with Percoll density gradient centrifugation and cultured bone marrow charge mesenchymal stem cells pure Shandong University master's degree thesis high ability of cell proliferation in vitro when cells better uniformity, the method is simple; spmol / the L 5 nitric cell celecoxib is more suitable concentration induced bone marrow mesenchymal stem cells into cardiomyocyte-like cell differentiation; immunofluorescence visible a Aetin, eardiae troponinl (eTnl)-positive cells were observed under transmission electron microscope to the part of the cell to the formation of actin filament-like structures , can be seen in the cells rich in mitochondria and glycogen granules. Conclusion: 1 whole bone marrow culture method is a simple, effective separation of mesenchymal stem cells in the bone marrow; of 2 spmol / L 5 nitric the cellular camp for the induction of bone marrow mesenchymal stem cells into cardiomyocyte-like cells to differentiate more appropriate concentration ; 3 bone marrow mesenchymal stem cells have more than can be differentiated characteristics, in vitro bone marrow mesenchymal stem cells can be in the 5-nitrogen cell celecoxib induced with a myocardial cell structure characteristics of cardiomyocyte-like cells differentiation, myocardial cell transplantation and lay a solid cell foundation . Keywords: bone marrow mesenchymal stem cells, cardiomyocytes, bone marrow culture method, cell differentiation, 5 nitric the Cellular \u0026 P, immunofluorescence surgery

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