体外定向诱导胚胎干细胞和诱导性多能干细胞分化为神经样细胞

发布时间：2018-12-15 11:23

【摘要】：视黄酸(Retinoic acid,RA)在神经系统中起着许多关键作用,例如神经分化,轴突伸长和神经模式的建成。在过去的几十年里,科学家发表了很多关于RA促进胚胎干细胞(embryonic stem cells,ESCs)分化形成神经细胞的研究论文,这些体外诱导实验通常涉及类胚体(embryoid bodys,EBs)的形成。我们现在报道的体外诱导ESC系R1分化形成神经细胞的方法是在单层贴壁培养系统中引进RA来实现的。诱导R1分化形成神经细胞的最初两天内,在N2B27培养基中加入1 ?μM的RA,可以提高ESCs向神经细胞分化的效率,具体表现在神经特异性的标记基因Sox1、Pax6、Nestin、Tuj1和MAP2表达水平的增加,以及Nestin、Tuj1和MAP2阳性的细胞数量的增多。另外,RA的添加还可以促进新生的神经元突出增长。我们的研究结果还显示:在神经发生的过程中, RA添加之后会伴随着WNT信号通路抑制因子Dickkopf-1(Dkk-1)表达水平的增加。直到目前为止,RA促进神经发生的机制仍不明确。单层贴壁培养系统不必牵涉复杂的EBs和不明因素的基质细胞,所以这一系统将能够更好利用到体外研究RA信号通路在神经发生过程中的作用上。诱导的多能干细胞(induced pluripotent stem cells,iPSCs)和ESC系R1一样,能够通过单层贴壁培养系统成功诱导产生神经细胞。实验中,我们选取了两种不同体细胞重编程产生的iPSCs作为研究对象,分别是来源于脑膜的iPSC克隆C5和来源于小鼠胚胎成纤维细胞(mouse embryonic fibroblasts,MEFs)的iPSC克隆S103f9。经过6天的诱导,两个iPSCs均能够检测到神经特异性的标记Pax6、Nestin、Tuj1和MAP2的表达,相反此时几乎不能够检测到多能性的标记Oct4的表达。这些结果与单层贴壁培养系统中诱导R1细胞神经发生时的状况一致,暗示了iPSCs与ESCs具有相似的神经发生能力。体外诱导病人自身的iPSCs形成神经细胞,可以作为研究某些神经系统遗传性疾病的模型,也能够为由于各种机能紊乱或年龄因素造成的神经组织的缺损提供移植治疗的材料。
[Abstract]:Retinoic acid (Retinoic acid,RA) plays a key role in the nervous system, such as neural differentiation, axon elongation and neural model formation. In the past few decades, scientists have published many research papers on RA promoting the differentiation of embryonic stem cells (embryonic stem cells,ESCs) into neurons. These in vitro induction experiments usually involve the formation of embryoid bodies (embryoid bodys,EBs). The method of inducing ESC R1 to differentiate into neurons in vitro was reported by introducing RA into monolayer adherent culture system. In the first two days of inducing R1 differentiation to form nerve cells, adding 1? 渭 M RA, to N2B27 medium can improve the efficiency of ESCs differentiation into nerve cells, as shown by the neural specific marker gene Sox1,Pax6,Nestin,. The expression of Tuj1 and MAP2 increased, and the number of Nestin,Tuj1 and MAP2 positive cells increased. In addition, the addition of RA can also promote the prominent growth of new neurons. Our results also showed that the addition of RA was accompanied by an increase in the expression of WNT signaling pathway inhibitor Dickkopf-1 (Dkk-1) during neurogenesis. Up to now, the mechanism of RA promoting neurogenesis is still unclear. The monolayer adherent culture system does not involve complex EBs and unknown stromal cells, so this system will be able to make better use of the in vitro study of the role of RA signaling pathway in neurogenesis. The induced pluripotent stem cells (induced pluripotent stem cells,iPSCs), like ESC strain R1, were successfully induced to produce nerve cells by monolayer adherent culture system. In the experiment, we selected two kinds of iPSCs from different somatic cell reprogramming, namely iPSC clone C5 derived from meninges and iPSC clone S103f9 from mouse embryonic fibroblast (mouse embryonic fibroblasts,MEFs. After 6 days of induction, both iPSCs could detect the expression of neuron-specific labeled Pax6,Nestin,Tuj1 and MAP2, whereas the expression of pluripotent labeled Oct4 could hardly be detected at this time. These results are consistent with those in monolayer adherent culture system, suggesting that iPSCs and ESCs have similar neurogenesis. Inducing iPSCs of patient to form nerve cells in vitro can be used as a model for studying some hereditary diseases of nervous system and as a material for transplantation treatment of nerve tissue defects caused by various functional disorders or age factors.
【学位授予单位】：中国科学技术大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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