| Bone marrow-derived mesenchymal stem cells (mesenchymal stem cell, MSCs) are present in the bone marrow, mesoderm and development of the Sino-a class of non-hematopoietic stem cells with multiple differentiation potential, not only can differentiate into hearntopoietic cells, can differentiate into a variety of blood outside the tissue cells, especially in mesoderm and neuroectodermal origin cells. MSCs in a number of areas, such as cell therapy, tissue engineering, developmental biology, gene function research, drug screening and other areas show a great prospect.Object:The experimental studies the characteristics of the bone marrow mesenchymal stem cells isolation,cultivation and expansion in vitro, The establishment of SD rat bone marrow mesenchymal stem cells cultured in vitro system.Method:1. Primary culture, Primary culture of rat MSCs were collected from the femurs and tibias of one month old SD rat. The bone marrow was washed from marrow cavity with L-DMEM complete medium. After filtrated, the marrow suspension were collected and placed in a 25 cm2 flask. The culture were maintained at 370C 5% CO2 and saturated humidity. The non-adherent cells were removed by replacing the medium after 72 hours of culture and thereafter to replace the medium every 72 hour. When the cells had grown to confluence(about 14 days), they were passaged by 0.25% trysin and replated after dilution to 1:2 or 1:3. The passaged MSCs atached to plastic flask within 12 hours and reached confluence within 7 to 10 days.2. Capability of proliferation of rat MSCs, The 1th,4th,7th,10th and 13th generation MSCs were selected to be investigated their capability of proliferation curve and adherence rate. The result showed.3. The antigenic phenotypes of rat MSCs, The 4th generation cells were collected and their expressions CD90 and CD44 are positive, CD34 and CD45 are negative.Result:1. adherent screening method, through replacement of the liquid can be repeated for separation, purification of MSCs, the method is simple, convenient and effective.2. MSCs have a strong proliferation ability in vitro, former than The 10th generation have the same cell morphology and proliferation ability, with the increase in the number of passages, the ability of poliferatrion weakened and irregular changes in morphogenesis. Cell growth adherent of the generation of 1,4,7,10,13 is basically the same as the rate of change, no significant difference.3. The antigen characteristic of MSCs surface markers present diversity, rBMSC hematopoietic cells did not express surface antigens CD34, CD45, show that MSCs is non-hematopoietic cells; while express surface markers of CD90, CD44, show that the characteristics of MSCs is non-uniformity. It expresses the surface markers of the mesenchymal cells,endothelial cells and epidermal cell.Conclusion:The experiments using bone marrow culture method for direct isolation and culture of MSCs, this method is simple, the effect was reliable, and through the passage of several generations will be able to obtain amplification of high purity,high number of MSCs, meet the needs of tissue engineering experimental research.Object:1. Construction of pEGFP-N1-BDNF plasmid.2. Get to the stable expression of BDNF in MSCs, and transfected rat bone marrow mesenchymal stem cells (MSCs), and observed the expression of BDNF in the MSCs。Method:Using RT-PCR method from rat bone marrow mesenchymal stem cells amplified with Xho I, BamH I restriction sites of the BDNF gene fragment was cloned into the BDNF gene enhanced green fluorescent protein (EGFP) gene eukaryotic expression vector pEGFP-N1, the construct pEGFP-N 1-BDNF plasmid. Transfected by electricity will be pEGFP-N1-BDNF transfected into rat MSCs。Result:pEGFP-N1-BDNF successfully constructed the eukaryotic expression vector, observed under fluorescence microscope Microscopep EGFP-N1-BDNF expression vector was transfected into MSCs 12h post-BDNF fusion protein expression can be seen.Conclusion:1. Successful Construction of pEGFP-N1-BDNF plasmid.2. PEGFP-N1-BDNF plasmid eukaryotic cells can be transfected HEK293 cells show that BDNF-EGFP fusion protein eukaryotic expression plasmid can be expressed in eukaryotic cells.3. Through the electrical resistance screening transfection and stable expression we get to the BDNF in MSCs.Object:1. Respectively use of beta-Mercaptoethanol (β-ME) and basic fibroblast growth factor (bFGF) as an induction agent, Differentiate the MSCs in to neurons and astrocytes in vitro successlly. Discuss the mechanism ofβ-ME and bFGF to induce thedifferentiation of MSCs into neurons cell, provide experimental basis for spinal cord injury of cell transplantation treatment.2. Discuss the impact of BDNF gene-modified MSCs differentiated neurons。 Method:1. Differentiate MSCs into the neurons cells, The 4th generation rBMSC cells were collected, by 2×104/ml concentration were inoculated in 24-well plates until the cells reached 70%-80% fusion, addingβ-S-ethanol (β-ME) and basic fibroblast growth factor (bFGF) respectively, inverted phase contrast microscope for observation the changes of the cell morphology.2. Immunofluorescence identification of cells, using cells immunofluorescence method, after the 5h cells of these two groups to further test markers TuJ1 neurons expression, observe them under fluorescence microscope and photographed record.3. the positive rate of MSCs differentiate into neurons, Fluorescence microscope check 300 cells in a random, count the number of TuJ1-positive cells, testing the group differences at different time points, and each time point the differences between the groups.Result:1. adding inducer into 4th generation MSCs, after cells differentiate into neurons and astrocytes. Observed under visible light:the former cells smaller, often have a few short processes and a longer process, showing the typical bipolar, multipolar and pyramidal-shaped structure, strongger refractive index; latter cells larger, have cell processes and more rough, and some were stellate shape, flat on the dish. 2. cells immunofluorescence showed that after 5h differentiation, some TuJ1-positive cells, were neurons.3. Two groups of MSC-induced neural cells, the positive rate for the comparison, BDNF-MSCs into neuronal cells fraction was higher than that MSCs group, and has statistical significance。Conclusion:1. MSCs in vitro and bFGF in the induction of agents under the effect ofβ-ME differentiate into neuron-like cells.2. After BDNF gene modification can significantly improve the rate of MSCs differentiation into neuron-positive。...
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