Sonic Hedgehog对神经干细胞增殖与分化的影响

发布时间：2018-05-07 19:49

  本文选题：sonic + hedgehog　； 参考：《南方医科大学》2009年硕士论文

【摘要】： 神经干细胞(neural stem cells,NSCs)指能自我增殖和自我更新,并具有向神经元和胶质细胞分化潜能的细胞。在神经系统发育的过程中,神经干细胞大部分分化成胶质细胞而只有少部分分化为神经元。大量关于神经干细胞体外分化实验或体内移植后检测的结果也显示,在没有特殊干预的情况下,大部分神经干细胞都将分化为星形胶质细胞,而分化成神经元或少突胶质细胞的比率很小。然而,移植神经干细胞治疗中枢神经损伤的主要目的却是希望它能在分化后替代死亡或退化的神经元或少突胶质细胞,所以如何促进神经干细胞向神经元或少突胶质细胞分化已经成为目前大家重点关注的问题。 Sonic Hedgehog(SHH)是胚胎发育过程中由脊索产生的一种重要的发育调控因子,根据胚胎发育时期SHH的作用机制及目前的研究进展,我们设想SHH有可能会对神经干细胞的增殖与分化发生一定的作用,但是,迄今为止尚未见相关的报道。为此,本课题拟通过体外实验,探讨SHH对神经干细胞的增殖与分化的影响,为神经干细胞定向诱导分化研究及体内移植实验提供基础。 本课题分为两部分: 第一部分 目的:从大鼠胚胎端脑分离培养神经干细胞,并对所获得的神经干细胞进行特异性蛋白表达以及增殖与分化能力鉴定。 方法:从大鼠胚胎(E9.5d)端脑分离培养神经干细胞,用含碱性成纤维细胞生长因子和表皮生长因子的无血清培养液进行培养。第2代细胞采取荧光免疫细胞化学检测神经干细胞特异性标志物nestin的表达;在培养液中添加BrdU 3h后检测BrdU在神经干细胞中的表达;在含血清而无生长因子的分化条件培养液中培养7d后经βⅢ-tubulin、GFAP、Rip和nestin荧光免疫细胞化学检测细胞分化情况。 结果:体外培养的神经干细胞能快速增殖并形成神经球,其中的细胞均表达神经干细胞特异性标志物nestin。所获得的细胞能将BrdU结合到细胞核中;部分细胞能分化为神经元、星型胶质细胞和少突胶质细胞。 结论:我们获得的细胞在特异性标志物、增殖与分化能力等方面均符合神经干细胞的特性。 第二部分 目的:探讨sonic hedgehog(SHH)对神经干细胞增殖与分化的影响。 方法:在分别将SHH、维甲酸(retinoic acid,RA)或PBS添加入培养液的情况下,对第2代神经干细胞的增殖能力(BrdU阳性细胞核的百分率)、分化能力(βⅢ-tubulin、GFAP、Rip和nestin阳性细胞百分率)进行检测,用SPSS13.0做One-way ANOVA分析,组间多重比较方差齐性时用LSD方法,方差不齐时使用Dunnett T3方法。 结果:SHH组的BrdU、βⅢ-tubulin和Rip阳性率均显著高于RA组和PBS组,nestin阳性率显著低于RA组和PBS组,GFAP阳性率各组间无显著差异。 结论:SHH可以提高神经干细胞的增殖能力,并可促进其向神经元和少突胶质细胞分化。
[Abstract]:Neural stem cells (NSCs) are cells that can proliferate and renew themselves and have the potential to differentiate into neurons and glial cells. During the development of the nervous system, most neural stem cells differentiate into glial cells and only a few differentiate into neurons. Numerous studies of neural stem cell differentiation in vitro or after in vivo transplantation also show that most neural stem cells will differentiate into astrocytes without special intervention. The rate of differentiation into neurons or oligodendrocytes is small. However, the main purpose of transplantation of neural stem cells in the treatment of central nerve injury is to hope that it can replace dead or degenerated neurons or oligodendrocytes after differentiation. Therefore, how to promote the differentiation of neural stem cells into neurons or oligodendrocytes has become the focus of attention. Sonic Hedgehog is an important developmental regulator produced by the notochord during embryonic development. According to the mechanism of SHH during embryonic development and its current research progress, Hedgehog is one of the most important developmental regulators in embryonic development. We hypothesized that SHH might play a role in the proliferation and differentiation of neural stem cells. Therefore, this study aims to explore the effect of SHH on the proliferation and differentiation of neural stem cells in vitro, and to provide a basis for the study of neural stem cell differentiation in vitro and in vivo transplantation. This topic is divided into two parts: Part one Aim: to isolate and culture neural stem cells from rat embryonic terminal brain and to identify the specific protein expression, proliferation and differentiation ability of the obtained neural stem cells. Methods: neural stem cells (NSCs) were isolated and cultured from the rat embryonic stem cells (E9.5 d) and cultured in serum-free medium containing basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). The expression of nestin, a specific marker of neural stem cells, was detected by fluorescence immunocytochemistry in the second generation of cells, and the expression of BrdU in neural stem cells was detected after adding BrdU for 3 h. The differentiation of the cells was detected by fluorescence immunocytochemistry of 尾 -tubulin Gapp Rip and nestin after being cultured in the medium containing serum but without growth factor for 7 days. Results: neural stem cells cultured in vitro could proliferate rapidly and form neurospheres, and all of the cells expressed the specific marker of neural stem cells, Neisin. The obtained cells can bind BrdU to the nucleus, and some of the cells can differentiate into neurons, astrocytes and oligodendrocytes. Conclusion: the obtained cells accord with the characteristics of neural stem cells in specific markers, proliferation and differentiation. Part two Objective: to investigate the effect of sonic hedgehog on the proliferation and differentiation of neural stem cells. Methods: under the condition of adding SHH, retinoic acid PBS or PBS into the culture medium, the percentage of BrdU positive nuclei and the differentiation ability (尾 鈪，

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