人胚胎干细胞向神经上皮祖细胞和多巴胺能神经元诱导分化的研究

发布时间：2018-06-04 04:20

本文选题：神经上皮祖细胞 + 饲养层细胞　；参考：《中南大学》2010年博士论文

【摘要】： 人胚胎干细胞(human embryonic stem cells, hESCs)从植入前囊胚的内细胞团(inner cell mass, ICM)分离而来,具有多向分化潜能和无限自我更新能力,是细胞替代治疗潜在的种子细胞来源,也为人类胚胎发育机制和基因功能研究及药物筛选等提供了新的材料。神经退行性病由于神经细胞的缺失或死亡后机体无法再生出新的神经细胞替代原有细胞的功能,如帕金森病、阿尔茨海默病和亨廷顿病等,发病率高,病程缓慢,临床上尚无有效的治疗手段,hESCs来源的神经细胞替代治疗可能会为这些难治性疾病提供新的治疗前景。 hESCs作为种子细胞要发挥作用,首先要建立将其定向诱导分化为特定细胞的诱导方法,在体外高效地诱导产生高纯度的特定神经细胞,并具备相应的生物学功能。目前,hESCs诱导为神经上皮细胞和神经元的方法各有千秋,但是相互之间缺乏系统比较。本研究采用本所建系的多株hESCs进行体外诱导,建立了一个简单、高效、无血清、无动物细胞污染的神经上皮祖细胞(neuroepithelial progenitors,NEP)和多巴胺能神经元(dopaminergic neurons, DAN)的诱导体系,为研究神经系统的早期发育机制和帕金森病细胞替代治疗提供相应的技术平台和研究材料。论文第一章的研究内容是建立简单高效的hESCs向神经上皮祖细胞诱导体系,比较发现人胚胎成纤维细胞(human embryonic fibroblasts, HEF)诱导体系与小鼠胚胎成纤维细胞(mouse embryonicfibroblasts, MEF)体系一样,可以在无血清培养条件下使hESCs直接贴壁诱导,产生具有典型rosette结构、表达Musashi, Nestin, Pax6等特异性抗原的NEP；且HEF体系的诱导效率更高。与悬浮诱导方法相比,直接贴壁诱导方法的效率更高。诱导培养基中添加或不添加外源性的成纤维细胞生长因子2(fibroblast growth factor 2, FGF2),对于NEP的诱导效率没有影响。采用无饲养层(feeder free, FF)可直接诱导出表达Musashi, Nestin和Pax6的NEP,及34%表达SSEA4的细胞。HEF和FF诱导体系下骨形成蛋白(bone morphogenetic protein, BMP)信号通路相关基因的检测显示,FF体系中BMP4高表达,而HEF中几无表达,提示HEF体系低BMP信号通路活性更有利于NEP的诱导发生。论文第二章的研究内容是建立HEF和FF体系产生的NEP的增殖扩增和向DAN诱导分化的体系。FGF2可以促进神经球和贴壁的NEP的增殖扩增,且其促进作用与FGF2呈浓度依赖性关系。NEP可在体外传6代以上,并维持相应NEP标记及向神经元和胶质细胞分化的潜能。在区域特化因子成纤维细胞生长因子8(fibroblast growth factor8,FGF8)和音猬因子(sonic hedgehog, SHH)的作用下,NEP可诱导分化为表达Otx2和Enl的中脑DAN前体,并进一步分化为酪氨酸羟化酶(tyrosine hydroxylase, TH)阳性的DAN。HEF和FF诱导分化终末阶段的产生的Tuj1和TH阳性细胞间没有差异,且没有SSEA4阳性细胞残留,说明两种体系下产生DAN的效率相同。FF体系不依赖于饲养层细胞,有利于神经发育生物学的研究及将来神经细胞诱导体系的产业化。论文第三章的研究内容是研究孤雌人胚胎干细胞(parthenogenetic human embryonic stem cells, phESCs)向NEP和DAN诱导分化的效率和安全性问题。和正常人胚胎干细胞(normal human embryonic stem cells, nhESCs)一样,phESCs可产生的Enl,TH和Tuj1阳性细胞,提示phESCs具备相同的向DAN分化的能力。NEP的比较染色体杂交检测未发现基因组染色体拷贝数的改变。印记基因H19, IGF2和Snrpn表达的检测发现,NEP基本维持了印记基因的表达模式。由此认为,phESCs具备向NEP和DAN分化的能力,体外分化中维持了其染色体和印记基因表达的稳定性,可以作为细胞替代治疗的来源。本研究通过上述结果证实,HEF体系是hESCs向NEP分化的高效诱导体系；FGF和BMP信号通路对于NEP的诱导起关键作用；FF体系获得的NEP可分化为DAN; phESCs具备正常的神经细胞分化潜能。我们建立的NEP和DAN诱导体系,为hESCs的临床和基础研究奠定了基础,为未来的神经细胞替代治疗提供了可能。
[Abstract]:Human embryonic stem cells ( hESCs ) are isolated from the inner cell mass ( ICM ) implanted into the blastocyst . It is a potential source of differentiation potential and unlimited self - renewal . It is a potential source of cell replacement therapy and provides new material for human embryonic development mechanism and gene function research and drug screening .

In vitro induction of hESCs , a simple , efficient , serum - free , non - animal cell - contaminated neuroepithelial progenitor cell ( NEP ) and dopaminergic neurons ( DAN ) were established to provide the corresponding technical platform and research material for the study of early development mechanism of nervous system and replacement therapy of Parkinson ' s disease cells .

The first part of this paper is to establish a simple and efficient induction system of hESCs to neural epithelial progenitor cells . It is found that the human embryonic fibroblasts ( HEF ) - induced system is the same as mouse embryonic fibroblasts ( MEFs ) system .
Compared with the suspension induction method , the efficiency of the direct adherent induction method is higher . The induction efficiency of the direct adherent induction method is higher . The induction efficiency of NEP is not affected by the addition or addition of exogenous fibroblast growth factor 2 ( FGF2 ) in the induction culture medium .

In the second chapter , the proliferation and amplification of NEP produced by HEF and FF systems were established .

In the third chapter , we study the efficiency and safety of the differentiation of genetic human embryonic stem cells ( phESCs ) to NEP and DAN . In normal human embryonic stem cells ( nhESCs ) , phESCs can produce Enl , TH and Tuj1 - positive cells , suggesting that phESCs has the same ability to differentiate the DNA .

In this study , the HEF system is an efficient induction system for the differentiation of hESCs to NEP .
FGF and BMP signaling pathways play a key role in the induction of NEP ;
The NEP obtained by FF system can be differentiated into DAN ; phESCs has normal nerve cell differentiation potential . We established NEP and DAN inducing system , which laid the foundation for clinical and basic research of hESCs , and provided the possibility for future neural cell replacement therapy .
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329

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