The Experimental Study Of Bone Marrow Stromal Cells Differentiated To Neuron Like Cells By NT3 Associated With TGF-β In Vitro

ObjectiveBone marrow stromal cells ( BMSCs) are a certain kind of stem cells different from haematopoietic stem cells in the marrow. BMSCs are an important composing part for the proliferation of haematopoietic stem cells. BMSCs have the a-bility of renovaton and differentiation themselves, BMSCs can differentiated to many organizations of ectoderm and mesoblast, form the bone,cartilage, muscle, fat and nerve et al. Under the special condition BMSCs can transform to the neural stem cells,and differentiated to astrocyte,oligodendrocyte and neuron et al. Researchs have found that the injected BMSCs can migrated into ischemic brain and differentiate into neuron like cells or glial cells, and BMSCs may secrete sorts of cytoking, which have important role in the brain injury reparative process. Then the investigation and application of neural stem cells differentiated from bone marrow turn into possible. There may be great clinic signification to discover the mechanism of differentiation in vitro. The experiment condition of differentiated to neuron like cells have not mature yet. The rate of differentiation still on a low level. The aim of the investigation is to study the isolateion, purification, amplification and biological character of BMSCs from rat bone marrow, To study the possibility of BMSCs differentiated into neural like cells. BMSCs could differentiate to neuron like cell which expressing NSE located on the cyto-plast. The whole experiments consists of isolation, puring, proliferation and differentiation of BMSCs in vitro, investigate the effect of differentiation by NT3 ( Neurotrophic factors 3 ) associated with TGF - β ( Transforming growth factor -β ). so as to provide the parameter and evidence for the clinic application of BMSCs in the future.MethodsThe original isolation, cultivation, identify and propagating and inducement. Including thereafter. The BMSCs were isolated from the thighbone, shank bone of Wistar rats of 6 weeks under axenic condition, being isolated by Percoll liquid, 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 lOOml/L fetal bovine serum. There were purified by passage control and adhering to the culture plastic. The BMSCs from adult rats were isolated and cultured for 6 passages. We observed the growth status of, and identify the phenotype of cells by immunocyto-chemistry of CD34 and CD44. We assume to using three different methods to induce the BMSCs to neuron like cells, in order to find a best method to inducing the bone marrow stromal cells. The 1st contrast group: The pre - liquid of inducement has no either TGF - (3 (Transforming growth factor - (3) NNT3 (Neu-rotrophic factors 3);The 2nd contrast group: The preliquid of inducement has TGF - (3 with ljxg/L only;The 3rd contrast group: The preliquid of inducement has NT3 with 50|xg/L. Then observed the morphology, and immunocytochemis-try, flow cytometry analysis were performed to calculate proportion of the positive cells of Nestin A NSE N (3 - catenin, and check the rate of differentiation at different point. Flow cytometry analysis were performed to calculate proportion of the positive cells of NSE, and check the rate of differentiation at different point by means of the second group experiment.Result1. We cultivated the BMSCs (bone marrow stromal cells) of passage one and the followed passage successfully, The BMSCs of passage one were oval, short spindle - shaped, or polygonal under the converted microscope. After pur-ification and amplification, they were uniform long spindle - shaped morphology. The living behavior of BMSCs from passage two to passage five 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. BMSCs were passaged every seven days. The proliferated abilty became weak after ten passages. The cytoplast become e-ven flat. If we add bFGF ( Basic fibroblast growth factor) , the morphology and proliferated abilty can be maintained. The livingness of BMSCs was all above 96% , 2 hours after passage there are 20 % cells adhere the cliff, with the time go on, the rate of adhibit was 98%.2. The special mark antigen of CD34( - ) ,CD44( + )were detected and positive express by immunocytochemistry.3. The BMSCs of contrast group have no any transformation, and do not express Nestin. The first group of experiment, a few BMSCs differentiated into neural stem cells, and express Nestin so faintly. The second group of experiment, about ten percent cells differentiated into neural stem cells and express Nestin at the same time and differentiated to neuron like cells which expressed NSE positive.The average positively rate of NSE and p - catenin was about 72. 45 +2. 05% , the 1st contrast group do not expressed NSE or(3 -catenin, the 2nd contrast group expressed NSE and (5 - catenin with a low level( <5% ) , the positive rate of 3rd group expressed NSE at about 48.02 + 1. 56%.4. Flow cytometry analysis : The rate of 12h of the differentiation higher than the others.ConclusionUnder the condition of NT3 associated with TGF - (3, The bone marrow stromal cells could differentiated to neural stem cells and differentiated neuron like cells at last.NT3 has the cooperate function with TGF - (3, which can improve the trans-form rate to the neuron like cells.