大胚龄人脑神经干细胞的分离培养及诱导分化的研究

发布时间：2018-01-10 17:32

本文关键词：大胚龄人脑神经干细胞的分离培养及诱导分化的研究　出处：《南昌大学》2006年硕士论文　论文类型：学位论文

【摘要】：目的 1．探讨大胚龄人脑神经干细胞(neural stem cells，NSCs)的分离及培养方法。 2．探讨骨髓基质细胞(bone marrow stromal cells，，BMSCs)对NSCs分化的影响。方法 1．从16-20周胚龄胎脑室下区分离组织，采用机械法获得单细胞悬液，台盼蓝染色计数细胞存活率。 2．采用无血清培养基(DMEM／F_(12))，加入2％B_(27)、EGF和bFGF培养NSCs，倒置显微镜下观察细胞的增殖情况；采用机械法分离法传代NSCs；采用免疫细胞荧光法检测神经上皮干细胞蛋白(nestin)及分化后神经细胞抗原的表达。 3．采用密度梯度离心结合贴壁分离法分离、纯化人BMSCs。 4．将人BMSCs和NSCs在体外通过不同的方式混合培养，实验分为三组：自然分化组、直接接触混合培养组及Transwell共培养组。观察BMSCs对NSCs分化的影响。采用免疫细胞荧光法及Western blot法检测各组神经元和星形胶质细胞标志物的表达。结果 1．采用机械法从胎脑室下区获得的单细胞悬液，台盼蓝染色显示细胞存活率80％左右。 2．NSCs原代培养时，镜下见单个细胞呈贴壁生长。在生长因子EGF和bFGF的刺激下，2天后细胞分裂增殖形成3—5个细胞聚成的团块。随着团块直径增大，逐渐形成形态规则的神经球，并呈悬浮生长，球周边不断有分裂
[Abstract]:objective
1. to investigate the isolation and culture of neural stem cells (NSCs) of human brain stem cells (SSCs) of large embryo age.
2. to investigate the effect of bone marrow stromal cells (bone marrow stromal cells, BMSCs) on the differentiation of NSCs.
Method
1. weeks from 16-20 fetal subventricular zone tissue separation, to obtain a single cell suspension by mechanical method, trypan blue staining and cell survival rate.
2. a serum free medium (DMEM / F_ (12)), 2%B_ (27), adding EGF and bFGF in cultured NSCs cell proliferation, observed under inverted microscope; separation passage NSCs using mechanical method; detection of neuroepithelial stem cell protein by immunofluorescence (nestin) and differentiation of nerve cells after antigen the expression.
3. the purification of human BMSCs. by the separation of density gradient centrifugation and adherent separation method
4. the BMSCs and NSCs in vitro by mixed culture of different ways, were divided into three groups: natural differentiation group, direct contact co culture group and Transwell group were cultured. The effect of BMSCs on differentiation of NSCs. Using immunofluorescence method and Western blot method to detect the neuron and astrocyte marker expression.
Result
1. the single cell suspension obtained by mechanical method was obtained from the subventricular region of the fetal brain. Trypan blue staining showed that the cell survival rate was about 80%.
2.NSCs were cultured, microscopically, single cells were adherent growth. The growth factor EGF and bFGF under the stimulation of cell proliferation, 2 days after the formation of 3 - 5 cells together into clumps. With mass diameter increases, gradually formed the morphological rules of neurospheres, and growth in suspension, the ball around the time a split

【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329.2

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