鼠胚神经干细胞的体外培养及其定向分化的实验研究

发布时间：2017-12-28 07:46

本文关键词：鼠胚神经干细胞的体外培养及其定向分化的实验研究　出处：《南京医科大学》2006年硕士论文　论文类型：学位论文

【摘要】：本研究旨在观察肝细胞生长因子(HGF)、胰岛素样生长因子-1(IGF-1)和脑源性神经营养因子(BDNF)对鼠胚神经干细胞(neural stem cell，NSC)体外诱导分化的促进作用，以及检测内源性bHLH基因-MASH1、neuroD、neurogenin2(Ngn2)在神经干细胞分化过程中表达的动态变化，探索神经干细胞分化的一些内部分子机制。首先取孕14-17天的SD大鼠的胚胎脑组织，在体外分离培养得到神经干细胞后，分为两组，实验组以HGF、IGF-1、BDNF等作为诱导分化剂诱导其分化，而对照组仅加入胎牛血清(FBS)，以免疫组化的方法鉴定所获得的神经干细胞、神经元(neuron)、星型胶质细胞(gliocyte)和少突胶质细胞(oliodendrocyte)，以镜下直接计数和流式细胞仪的方法来计数诱导分化的神经元比例。选取未分化、分化1天、3天、5天做为观察和研究的时间段，采用RT-PCR的方法分析分化各个时期内源性bHLH基因表达的差异。原代培养结果显示：在培养基中生长的细胞不断出现形态学的改变，，4天左右，由一个细胞分裂而来的细胞已经长为几十个细胞聚集在一起的悬浮生长的神经克隆球，培养至1周左右时，神经球增多、增大，边缘可见到锯齿状的单细胞边界，形态规则，立体感强。诱导分化4-8小时后，两组在倒置显微镜下均可以观察到部分原来呈悬浮生长的克隆球贴壁，逐渐失去其球状的立体外观。24小时后即可发现从细胞团块的边缘向外长出突起，克隆球中的细胞有向外迁移的趋势，3天后即有不同形态的细胞从克隆球周围出现，5天后克隆球已完全附壁。Nestin是神经
[Abstract]:The purpose of this study was to observe the hepatocyte growth factor (HGF), insulin-like growth factor -1 (IGF-1) and brain-derived neurotrophic factor (BDNF) on rat embryonic neural stem cells (neural stem cell, NSC) promotes differentiation in vitro, and to detect endogenous bHLH genes -MASH1, neuroD, neurogenin2 (Ngn2) in the dynamic changes of the expression of the differentiation of neural stem cells into neural stem, some internal molecular mechanism of cell differentiation. The brain tissue of SD rat embryos from the first 14-17 days of pregnancy, in isolated neural stem cells, divided into two groups, the experimental group with HGF, IGF-1 and BDNF as the inducer of differentiation inducing differentiation, while the control group only adding fetal bovine serum (FBS), stem cell and neuron method identified by immunohistochemistry for nerve (neuron), astrocytes (gliocyte) and oligodendrocytes (oliodendrocyte), the proportion of neurons through direct counting under microscope and flow cytometric methods to count differentiation. 1 days, 3 days and 5 days were selected as observation and research period. The difference of endogenous bHLH gene expression at different stages was analyzed by RT-PCR. The results showed that primary cultured in DMEM cells appeared morphological changes, about 4 days from a cell division and the cell has been long for dozens of cells together suspended growth neural clone ball, cultured for 1 weeks or so, neurospheres increased and enlarged, single the cell boundary, the edge can be seen jagged shape, three-dimensional sense of strong. After induction and differentiation for 4-8 hours, two groups could observe some of the original cloned spheres attached to the suspension growth under the inverted microscope, and gradually lost their spherical appearance. 24 hours later, it was found that protuberance grew outward from the edge of the cell mass, and the cells in the cloned sphere migrated outward. After 3 days, there were different forms of cells from the clones, and 5 days later, the clones were completely attached to the wall. Nestin is the nerve
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329.2

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