| Part 1: Separated,cultured and expanded mesenchymal stem cells from rat bone marrow in vitroOBJECTIVE: To study the isolation, purification of rat bone marrow mesenchy mal stem cells (BMSCs) in vitro and compare cell expansion power under different inoculation density.METHODS: BMSCs were separated by density gradient centrifugation on Ficoll (density, 1.077×103g/L) and were purified by adhering to the culture plastic, then incubated with DMEM supplemented 10% fetal bovine serum (FBS) . After subcultured by three different inoculation density of 50000/cm2, 5000/cm2, and 500/cm2, observed the cell morphological changes, drawn the growth curve, and measured the doubling time in log phase by cell counting.RESULTS: After 24 hours on primary culture, some adherent cells were observed and the morphology changed from sphere into fusiform. The doubling time was 30.4h in 50000/cm2, 36.1h in 5000/cm2, and 38.2 h in 500/cm2. BMSCs exhibited thelargest expansion potential in inoculation density of 500/cm2.CONCLUSION: BMSCs could be effective isolated and purified by density gradient centrifugation and adhering to the culture plastic and expanded satisfactorily in DMEN with 10% FBS. When inoculating under low density, the expansion power of BMSCs is stronger comparably .Part 2: Induced BMSCs to differentiate into neuron-like cells with conditioned medium of neural stem cells in vitroOBJECTIVE: To investigate the differentiation from BMSCs into neuron-like cells induced with conditioned medium of neural stem cells.METHODS: BMSCs were induced with Conditioned medium collected in advance. Cells morphological changes were observed under phase contrast microscope, and the specific marking proteins, such as neural-specific enolase (NSE), Nestin and glial fibrillary acidic protein (GFAP) were detected by immunocytochemistry. Induced by DMSO and BHA as control group.RESULTS: In experiment group , 6 h after induction, some BMSCs were transformed into neuron-like cells , at 24 h, the number of survival neuron-like cells was 43±8, the percentage of NSE positive cells was 13±6, both of them were significantly higher than that of control group(P<0.05).CONCLUSION: BMSCs can be induced to differentiate into neuron-like cells in vitro successfully by conditioned medium of neural stem cells and the number of induced survival neuron-like cells is more than induced method of DMSO+BHAPart 3: Transplantation of induced BMSCs in a rat model oftraumatic brain injuryOBJECTIVE: To study the influence on neurological functional restoration and structural changes after transplanted the induced BMSCs into the rats subjected to traumatic brain injury.METHODS: BMSCs marked with BrdU and induced by conditioned medium of neural stem cells were injected into the injury region. The neurological function of the rats was evaluated using the neurological severity score(NSS) at 4d, 1w, 2w, and 4w after transplantation. The structural changes in injury region were observed by immunocytochemistry. At 2w and 4w after transplantation and the transplanted BMSCs migration in brain was detected through identifying BrdU-positive cells.RESULTS: Rats neurological functional were improved significantly at 2w and 4w after transplanting induced BMSCs; the scores ware 5.2±0.8, 2.1±0.8 respectively and compared other groups, these scores were all low dramatically (P<0.05).CONCLUSION: The method of transplanting induced BMSCs might contribute to restoration rat neurological function in traumatic brain injury. |
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