稳定表达转录因子FoxA1或FoxM1对干细胞多能性及其分化潜能影响的研究

发布时间：2018-03-31 02:33

本文选题：P19EC细胞　切入点：稳定细胞株　出处：《湖南大学》2011年硕士论文

【摘要】：干细胞是一类具有自我更新能力和多向分化增殖潜能的原始细胞。叉头匡蛋白家族是细胞内一类非常重要的转录因子,参与了细胞的新陈代谢,发育,分化,增殖,凋亡等几乎所有的生理过程。本文以P19EC细胞作为研究体系,重点研究了Fox家族的两个重要成员FoxA1和FoxM1。为了研究FoxA1对干细胞多能性及分化潜能的影响,我们构建了能在P19细胞中稳定表达GFP-rFoxA1融合蛋白的细胞株。通过分析发现,P19 GFPFoxA1细胞株能正常表达多能性关键基因如Oct4, Nanog和Sox2,但是它也能表达神经干细胞标志基因Nestin和神经特异性基因TubulinβIII。同时,我们检测到细胞株的碱性磷酸酶的活性明显下降,而阶段特异性胚胎抗原SSEA-3和神经干细胞相关膜蛋白Prominin-1出现了高表达。这些暗示了细胞株呈现部分神经干细胞的特征。在RA诱导P19细胞分化的过程中,P19 GFPFoxA1细胞株在分化的早期就能看到多能性基因Nanog的迅速下调,而TubulinβIII在分化的早期增加的更快些,暗示FoxA1的高表达可能可以促使细胞更早地启动分化。所以我们认为在P19细胞中高表达FoxA1,可能会使P19细胞变成类似于神经干细胞的细胞。本文还通过构建P19 GFPFoxM1稳定细胞株来探究FoxM1与细胞多能性维持的关系。通过分析发现,在RA诱导的分化的过程中,FoxM1的稳定高表达使细胞不易形成胚胎小体,多能性关键基因如Oct4、Sox2也能更持久的维持表达,说明FoxM1可能对维持干细胞的多能性起重要作用。
[Abstract]:Stem cells are a kind of primitive cells with the ability of self-renewal and multidirectional differentiation and proliferation. The family of fork head proteins is a very important transcriptional factor in cells involved in cell metabolism, development, differentiation and proliferation. In this paper, P19EC cells were used as the research system to study two important members of the Fox family, FoxA1 and FoxM1. In order to study the effect of FoxA1 on pluripotency and differentiation potential of stem cells, We have constructed a cell line that can stably express GFP-rFoxA1 fusion protein in P19 cell line. We found that P19 GFPFoxA1 cell line can normally express multipotent key genes such as Oct4, Nanog and Sox2, but it can also express neural stem cell marker gene Nestin. And neuro-specific gene Tubulin 尾 III. at the same time, We detected a significant decrease in alkaline phosphatase activity in cell lines. However, the high expression of stage specific embryonic antigen (SSEA-3) and neural stem cell associated membrane protein (Prominin-1) suggested that the cell line showed the characteristics of some neural stem cells. During the differentiation of P19 cells induced by RA, the P19 GFPFoxA1 cell line was expressed in the P19 cell line. The rapid down-regulation of the pluripotent gene Nanog can be seen early in differentiation. However, Tubulin 尾 III increased more rapidly at the early stage of differentiation, suggesting that the high expression of FoxA1 might induce cells to start differentiation earlier, so we think that overexpression of FoxA1 in P19 cells may make P19 cells become neural stem cell like cells. The relationship between FoxM1 and the maintenance of cellular pluripotency was explored by constructing P19 GFPFoxM1 stable cell line. It was found that the stable and high expression of FoxM1 made it difficult for cells to form embryosomes during the differentiation induced by RA. Multipotent key genes such as Oct4nSox2 can also be maintained more persistently, suggesting that FoxM1 may play an important role in maintaining pluripotency of stem cells.
【学位授予单位】：湖南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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