| These years, It was previously known that the investigation of stem cells have made great remarkable progress, The neural stem cells ( NSCs) have been the foucus issue in the international neurology field with the other name is neural precursor cells. They can differentiate into neuron , astrocyte and oligodendro-cyte in some conditions. As the transplanted seed cells and carrier cells, NSCs have extensively applications, NSCs may be the most available tools to cure the neuropathic disease such as Parkinson disease , demyelinate disease and central nerve system injury, such as cerebric injury and spinal cord injury et al. The neural stem cells which were transplanted into the injuried spinal cord could be detected being differented into astrocyte, oligodendrocyte and neuron et al. Reconstructed the neural conductive rute and improved the function and sense of the lower limbs. But the source of the cells and ethic restricted the application of clinic. In addition, even the weakest immune repulsive reaction would affect the survive of transplanted cells. The NSCs which derived of own - self can solve the problem, but the NSCs in the nerve system has the difficulty in extraction, which limits the clinic application. In order to solve this problem, many scientists apply theirselves to exploring one new way to obtain the NSCs. Therefore, The aim of the investigation is to study the isolateion, purification, amplification and biological character of mesenchymal stem cells (MSCs) from rat bone marrow. To study the possibility of MSCs differentiated into neural precursor cells ( neural stem cells). MSCs could differentiate to neural stem cell which expressing Nestin located on the cytoplast. The whole experiments consists of isolation, puring, proliferation and differentiation of MSCs, so as to provide the parameter and evidence for the clinic application of mesenchymal stem cells in the future.MethodsThe original isolation, cultivation, identify and propagating and inducement. Including thereafter the mesenchymal stem cells were isolated from the thighbone, shankbone of Wistar rats of 8 weeks under axenic condition, being i-solated by Percoll, so as to obtain one certain kind of simplex cells, According to the ability of affixing, filtrating the cells which were easy to affix and fall off to propagate. The original cells were culture in DMEM containing 100ml/L fetal bovine serum. There were purified by passage control and adhering to the culture plastic. The bone marrow mesenchymal stem cells from adult rats were isolated and cultured for 5 passages. We observed the growth status of, and identify the phenotype of cells by immunocytochemistry of CD45 and CD90. We assume to using three different methods to induce the mesenchymal stem cells to neural stem cells, in order to find a best method to inducing the cells. The contrast group: The preliquid of inducement has no bFGF (Basic fibroblast growth factor) EGF (Epidermal Growth Factor) and formal liquid has no BHA (buty-lated hydroxyanisole ) DMSO ( dimethylsulfoxide ) BDNF ( brain derived neurophic nutrient factor) either;The group one of experiment; The preliquid of inducement has bFGF EGF and no BHA DMSO BDNF; The group two of experiment: The preliquid of inducement has no bFGF EGF and the formal liquid has BHA DMSO BDNF. Then observed the morphology, and immunocytochemistry , flow cytometry analysis were performed to calculate proportion of the positive cells of Nestin, and check the rate of differentiation at different point.Result1. We cultivated the mesenchymal stem cells of passage one and the followed passage successfully, The mesenchymal stem cells of passage one were o-val, short spindle - shaped, or polygonal under the converted microscope. After purification and amplification, they were uniform long spindle - shaped morphology. The living behavior of mesenchymal stem cells from passage two to passagefive were quite stable. The cells of passage one welted on the 2nd day, becoming collection between the 5th day and 6th day, overspread on the 10th day. Exhibiting a large expansive potential under passage five. Mesenchymal stem cells were passaged every seven days. The proliferated ability became weak after ten passages. The cytoplast become even flat. If we add bFGF ( basic fibroblast growth factor) , the morphology and proliferated abilty can be maintained.2. The special mark antigen of CD45 ( - ) , CD90 ( + ) were detected and positive express by immunocytochemistry.3. The mesenchymal stem cells of contrast group have no any transformation , and do not express Nestin. The first group of experiment, a few mesenchymal stem cells differentiated into neural stem cells, and express Nestin faintly. The second group of experiment, about ten percent cells differentiated into neural stem cells and express Nestin at the same time, flow cytometry analysis were performed to calculate proportion of the positive cells of Nestin, and check the rate of differentiation at different point by means of the second group experiment. The average inducible rate of differentiation at the 5th hour is 49. 79% , and the average inducible rate of differentiation on the 1st day is 31. 91% , the average inducible rate of differentiation on the 6th day is 14. 43% , the fifth -rate is high. The induced cells showed different morphology compared to those of the MSCs, but similar to those of the neural stem cells.ConclusionThe mesenchymal stem cells could differentiated into neural precursor cells with the transformation of morphology and the expression of nestin, the neural precursor cells have the same character of neural stem cells. |
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