新生大鼠神经干细胞定向分化为胆碱能神经元的实验研究

发布时间：2018-04-06 01:34

本文选题：神经干细胞　切入点：新生大鼠　出处：《重庆医科大学》2007年硕士论文

【摘要】： 第一部分新生大鼠神经干细胞的分离、培养研究背景与目的神经干细胞(neural stem cell, NSC)是近年来神经科学领域的研究热点。NSC是具有自我更新能力和多向分化潜能的细胞,可分化为神经系统的神经元、星形胶质细胞和少突胶质细胞,在胚胎及成年哺乳类动物中广泛存在。NSC的分离培养是其基础和应用研究的前提,本部分目的在于获得新生大鼠NSCs,为深入研究其增殖分化奠定基础。方法从新生24h内大鼠端脑及中脑部位分离NSC,采用体外无血清悬浮培养,以获得能在体外长期生存的NSC;通过免疫细胞化学检测NSC标记物nestin表达及分化为神经元和胶质细胞的能力。结果获得的NSC能在体外长期生存,具有很强的增殖能力、自我更新能力,表达nestin,具有多向分化能力,能分化为神经元和胶质细胞。结论成功地从新生大鼠端脑和中脑中分离得到了能在体外长期培养的NSC,培养的NSC表达nestin,有自我更新、多向分化的特点,通过培养细胞大量扩增。第二部分ATRA、NGF诱导新生大鼠神经干细胞定向分化为胆碱能神经元的实验研究研究背景及目的神经干细胞为神经系统疾病细胞替代治疗提供了细胞来源,疾病的治疗需要大量特定表型和功能的神经细胞,因此,对NSC分化为某种特定表型的研究成为神经科学研究的焦点。阿尔茨海默病(AD)是一种以基底前脑胆碱能神经元的退变为主要病理特征的神经系统变性疾病,补充胆碱能神经元是治疗该疾病的一种策略。本部分的目的在于在体外环境下使NSC定向分化为胆碱能神经元,为深入研究NSC体外分化条件,探讨诱导分化机制,提高胆碱能神经元分化效能奠定细胞学基础。方法在第3代NSCs无血清培养体系中加入ATRA和/或NGF,分别对NSC克隆进行ATRA、NGF、ATRA联合NGF诱导分化,通过免疫细胞学方法检测胆碱能神经元标志物CHAT的表达,比较不同诱导因素对NSC定向分化为胆碱能神经元的作用。结果ATRA、NGF单独诱导均提高CHAT阳性细胞表达,与血清对照组比较有显著性差异(P0.05),而ATRA联合NGF诱导组与单独应用NGF组比较不能显著提高CHAT阳性细胞率,二者无显著性差异(P0.05)。结论ATRA和NGF均有诱导NSC向胆碱能神经元分化的作用,但两者并不存在协同诱导作用。
[Abstract]:Part 1 Isolation and culture of neural stem cells from neonatal rats
Background and objective neural stem cells (neural stem cell, NSC) is a hotspot in the field of neuroscience is.NSC with self-renewal and multilineage differentiation potential of cells can differentiate into neurons, astrocytes and oligodendrocytes in embryonic and adult mammalian animal widely existed in culture.NSC separation is the premise of basic and applied research. The objective of this part is to obtain the NSCs neonatal rat, which laid the foundation for the further study on its proliferation and differentiation.
Methods NSC was isolated from the midbrain and midbrain of neonatal 24h in rats, and serum-free suspension culture was used to obtain long-term NSC in vitro. The expression of NSC marker nestin and the ability to differentiate into neurons and glial cells were detected by immunocytochemistry.
Results NSC can survive in vitro for a long time. It has strong proliferative ability, self-renewal ability, and nestin expression. It has multiple differentiation ability and can differentiate into neurons and glial cells.
Conclusion NSC, which can be cultured in vitro for a long time, is successfully isolated from the midbrain and midbrain of neonatal rats. The NSC expression of nestin is self renewing and multidirectional. It is amplified by culture cells.
The second part ATRA and NGF induced the directional differentiation of neural stem cells into cholinergic neurons in neonatal rats
Background and objective neural stem cells provide a cell source for cell replacement therapy of nervous system diseases, disease treatment requires a lot of specific phenotype and function of nerve cells, therefore, to study the differentiation of NSC into a specific phenotype has become the focus of neuroscience research. Alzheimer's disease (AD) is a neurodegenerative disease in basal forebrain cholinergic neurons degeneration is the main pathological features of cholinergic neurons, is a kind of strategy in the treatment of the disease. The purpose of this section is in vitro under the environment of NSC to differentiate into cholinergic neurons, for further research on NSC in vitro differentiation conditions, explore the mechanism of inducing differentiation, improve cholinergic cell basis for the neuronal differentiation efficiency.
Methods with ATRA and / or NGF system in serum culture in the third generation NSCs, respectively NSC clones were ATRA, NGF, ATRA and NGF to induce differentiation, by immunocytologic detection method of cholinergic expression of neuronal markers CHAT, comparison of different factors on NSC induced differentiation into cholinergic neurons.
Results ATRA and NGF alone increased the expression of CHAT positive cells. There was a significant difference compared with the serum control group (P0.05), while the ATRA plus NGF induction group did not significantly increase the CHAT positive cells rate compared with the NGF group alone, but there was no significant difference between the two groups (P0.05).
Conclusion both ATRA and NGF can induce the differentiation of NSC to cholinergic neurons, but there is no synergistic effect between them.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329.28

【引证文献】