大鼠神经干细胞的体外培养及向神经元的诱导分化

发布时间：2018-08-13 19:16

【摘要】：神经干细胞的发现,更新了以往认为神经元不能再生的传统观点,使人们对神经系统的结构和功能有了一个全新的认识。神经干细胞不仅可以作为研究神经发育的模型,并且,作为潜在的移植细胞的来源,为临床治疗神经系统疾患提供了一个新的有效治疗途径。如果神经干细胞能像造血干细胞那样得到深入研究,并且能够大量获得,进而异体移植成功,必然会对医学进步起重大的推动作用。目前,关于神经干细胞的研究虽然很多,但距离实际应用还有很大的差距。因此,选择神经干细胞作为研究对象,对体外培养的神经干细胞的生长特性,形态结构进行较深入的研究;并探讨诱导神经干细胞向神经元定向分化的有效方法;同时检测神经干细胞中DNA 拓扑异构酶II 的表达情况,期望能够找到调控神经干细胞增殖和定向分化的新切入点。本研究分四部分。第一部分:大鼠神经干细胞体外生长特性目的:介绍一种较成熟的大鼠神经干细胞体外培养方法,并观察其体外生长特性。方法: 1 神经干细胞的分离培养取新生大鼠(1d 之内)侧脑室前角周围的脑组织,用EDTA㑳胰蛋白酶(1㑳1)消化, 制成单细胞悬液,接种于无血清培养基(含B27 和20ng/ml bFGF,20ng/ml EGF 的DMEM/F12 培养基)中,观察神经干细胞的体外生长过程,并比较不同接种密度,不同传代时间对神经干细胞生长的影响。2 大鼠神经干细胞的鉴定2.1 nestin 免疫细胞化学:取传至第3 代细胞,传代后3d,做nestin 免疫细胞化学,一抗为兔抗鼠nestin 多克隆抗体(1:50),4℃,24h;FITC标记的二抗(1:30),37℃,2h。2.2 生长曲线绘制:取传至第3 代的神经干细胞,以5×10~5个细胞/ml 密
[Abstract]:The discovery of neural stem cells has renewed the traditional view that neurons can not regenerate, and has given people a new understanding of the structure and function of the nervous system. If neural stem cells can be thoroughly studied like hematopoietic stem cells, and can be obtained in large quantities, and then allogeneic transplantation is successful, it will certainly play a major role in promoting medical progress. To study the growth characteristics and morphology of neural stem cells cultured in vitro, explore the effective methods of inducing neural stem cells to differentiate into neurons, and detect the expression of DNA topoisomerase II in neural stem cells, hoping to find a way to regulate neural stem cells. The new breakthrough point of cell proliferation and directional differentiation is divided into four parts.
Part one: growth characteristics of rat neural stem cells in vitro
OBJECTIVE: To introduce a mature method for culturing rat neural stem cells in vitro and observe their growth characteristics.
Method:
1 Isolation and culture of neural stem cells
The brain tissue around the anterior horn of lateral ventricle of neonatal rats (within one day) was digested with EDTA? Trypsin (1? 1) and made into a single cell suspension. The cells were inoculated in serum-free medium (DMEM / F12 medium containing B27 and 20ng / ml bFGF, 20ng / ml EGF). The growth process of neural stem cells in vitro was observed and the effects of different inoculation densities and passages on the nervous system were compared. Identification of neural stem cells by stem cell growth. 2. 1 nestin immunocytochemistry: 3 days after passage, nestin immunocytochemistry, rabbit anti-nestin polyclonal antibody (1:50), 4 C, 24 h; FITC labeled second antibody (1:30), 37 C, 2 h. The cells were 5 * 10~5 cells /ml dense.
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R329

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