Nanog在胚胎干细胞自我更新中的作用机制研究

发布时间：2018-07-17 19:35

【摘要】： 胚胎干细胞(embryonic stem cells,ES cells)是一群具有多向分化潜能的细胞,发育上的全能性赋予其在临床上广泛的应用前景。对胚胎干细胞全能性机制的研究是其在临床上得以应用的前提和基础。Nanog是2003年新发现的一个转录因子,特异性的表达于全能性细胞中,研究发现Nanog在胚胎干细胞的全能性维持、早期分化以及成体细胞的去分化过程中起至关重要的作用,但是其具体的作用机制现在仍未阐明。本研究旨在通过研究Nanog的下游靶基因、上游信号转导通路调控、以及其是否参与成体细胞的去分化过程来阐明Nanog在胚胎干细胞自我更新中的作用机制。本研究的第一部分旨在研究胚胎干细胞中受Nanog表达调控的相关基因。Nanog作为一个转录因子通过调控下游基因的表达来发挥作用,对这些基因的分析研究能够更好地了解Nanog在胚胎干细胞中的作用机制。我们采用RNA干扰(RNA interference,RNAi)的方法特异性的降低胚胎干细胞中Nanog的表达,为了得到有效的干扰片断,我们设计合成了四个特异性的Nanog干扰片断以及一个阴性对照片断。从中筛选得到一个高效的干扰片断Nanog-siRNA-P1,Western-Blot和Real-Time PCR分析结果显示Nanog-siRNA-P1对Nanog表达的抑制效率可达90%。Nanog表达降低后,胚胎干细胞呈现分化特征,并伴随着细胞增殖能力下降。在用RNA干扰方法沉默Nanog表达24h和48h后,采用表达谱芯片筛选变化基因。我们认为这些差异表达基因为Nanog的相关下游靶基因,对这些基因的进一步研究分析将有助于我们了解Nanog在ES细胞全能性维持中的作用机制。在本研究的第二部分中,我们观察了PI3K信号转导通路对胚胎干细胞自我更新能力以及Nanog表达的影响。首先,我们观察到利用PI3K信号通路特异性的抑制剂LY294002特异性地抑制PI3K通路的激活后,ES细胞呈现明显的分化状态,Nanog表达下降。为了了解PI3K信号通路在ES细胞自我更新中的作用是否是通过调节Nanog的表达介导的,我们构建了Nanog-GFP融合蛋白表达载体,并转染ES细胞,G418筛选后得到过表达Nanog的ES细胞(Ex-Nanog-J1)。结果表明Nanog的过表达可以使小鼠的ES细胞在无LIF条件下保持不分化状态,并可以减弱由于LY294002所引起的ES细胞分化,但是外源性的Nanog并不能完全逆转这一分化作用。本研究的第三部分中我们观察了Nanog在成体细胞去分化中的作用。大家都知道胚胎干细胞可以在适宜的条件下向三个胚层的各种组织细胞分化,成体细胞也可以通过细胞融合(cell fusion)以及成体细胞核移植(somatic cells nucleartransfer,SCNT)的方法去分化到多能性干细胞阶段。Nanog特异性的表达于胚胎干细胞中,并且在胚胎干细胞的多能性维持中也起重要作用,那么,Nanog是否参与成体细胞的去分化过程呢?为了研究Nanog在成体细胞去分化中的作用,我们构建了Nanog-GFP融合蛋白表达载体,并转染3T3细胞,G418筛选得到表达Nanog的3T3细胞,观察到外源性Nanog特异性的表达于3T3细胞的细胞核中,这与Nanog在胚胎干细胞中的表达定位相同。继而我们观察了表达Nanog的3T3细胞是否向着全能性细胞转化,镜下观察其细胞形态并没有显著的变化,细胞周期检测Nanog可以提高3T3细胞的S期降低G0/G1期和M期,可以激活Oct4的表达,但是却未检测到其它的ES细胞相关基因Sox2、Utf1、Ets2的表达。这些表明Nanog在成体细胞去分化过程中,可能会起一定的作用,但是单纯的Nanog过表达并不足以使成体细胞去分化到全能性细胞阶段。通过该项研究我们发现:Nanog在胚胎干细胞的自我更新过程中起至关重要的作用,作为一个转录因子Nanog通过促进全能性相关基因的表达以及抑制分化相关基因的表达来维持ES细胞的不分化状态;PI3K信号通路参与胚胎干细胞全能性的维持,利用LY294002(PI3K通路特异性的抑制剂)抑制PI3K通路后诱导ES细胞分化。外源性地过表达Nanog会削弱由于LY294002引起的分化,但是却不能完全逆转这一分化作用,这表明PI3K信号通路在ES细胞中的作用只有一部分是通过调节Nanog的表达来介导;Nanog在成体细胞的去分化过程中起一定的作用,但是单纯的Nanog过表达并不足以使成体细胞去分化到全能性干细胞阶段。
[Abstract]:Embryonic stem cells (ES cells) is a group of cells with pluripotent differentiation potential. The developmental omnipotency gives it a wide range of clinical applications. The research on the omnipotent mechanism of embryonic stem cells is the premise and base of its clinical application and the.Nanog is a newly discovered transcription factor in 2003. The study found that Nanog plays an important role in the omnipotent maintenance, early differentiation and dedifferentiation of embryonic stem cells, but its specific mechanism is still not elucidated. The aim of this study is to study the target gene of Nanog, the regulation of the upstream signal transduction pathway and the regulation of the upstream signal transduction pathway. Whether it participates in the dedifferentiation process of adult cells to elucidate the mechanism of Nanog in the self-renewal of embryonic stem cells.
The first part of this study aims to study the role of.Nanog, a related gene regulated by Nanog expression in embryonic stem cells as a transcription factor, to play a role in regulating the expression of downstream genes. The analysis and research of these genes can better understand the mechanism of Nanog in the embryonic stem cells. We use RNA interference (RNA interference). RNAi's method specifically reduced the expression of Nanog in embryonic stem cells. In order to get an effective interference fragment, we designed and synthesized four specific Nanog interference fragments and a negative control fragment. A highly efficient interference fragment Nanog-siRNA-P1 was screened from it, and the Western-Blot and Real-Time PCR analysis results showed Nanog-. The inhibition efficiency of siRNA-P1 on the expression of Nanog could reach the decrease of 90%.Nanog expression, and the embryonic stem cells showed differentiation characteristics and decreased with the cell proliferation ability. The expression gene chip was used to screen the change genes after Nanog expression of 24h and 48h with RNA interference method. We think these differentially expressed genes are the related downstream target genes of Nanog. Further analysis of these genes will help us understand the mechanism of Nanog in the maintenance of ES cell totipotency.
In the second part of this study, we observed the effect of PI3K signal transduction pathway on the self-renewal capacity of embryonic stem cells and the expression of Nanog. First, we observed that ES cells showed distinct differentiation and Nanog expression decreased after the specific inhibition of the activation of the PI3K pathway by the specific inhibitor LY294002 of the PI3K signaling pathway. In order to understand whether the role of PI3K signaling pathway in ES cell self renewal is mediated by regulating the expression of Nanog, we constructed a Nanog-GFP fusion protein expression vector and transfected ES cells. G418 screened the ES cells expressing Nanog (Ex-Nanog-J1) after G418 screening. The results showed that the overexpression of Nanog could make ES cells in mice without LIF. The condition remained undifferentiated and weakened the differentiation of ES cells induced by LY294002, but exogenous Nanog did not completely reverse this differentiation.
In the third part of this study, we observed the role of Nanog in the dedifferentiation of adult cells. It is known that embryonic stem cells can differentiate into various tissue cells of the three germ layers under suitable conditions, and adult cells can also pass cell fusion (cell fusion) and adult somatic cells nucleartransfer (SCNT). .Nanog specifically expresses in embryonic stem cells and plays an important role in the pluripotent maintenance of embryonic stem cells. Then, does Nanog participate in the dedifferentiation process of adult cells? In order to study the role of Nanog in the dedifferentiation of adult cells, we constructed a Nanog-GFP fusion. Protein expression vector, and transfection of 3T3 cells, G418 screening 3T3 cells expressing Nanog, the expression of exogenous Nanog specifically expressed in the nucleus of 3T3 cells, which is the same as the expression of Nanog in the embryonic stem cells. There is no significant change in cell morphology. Cell cycle detection Nanog can increase the S phase of 3T3 cells to decrease G0/G1 and M phase, and can activate Oct4 expression, but no other ES cell related genes Sox2, Utf1, Ets2 expression are not detected. These indicate that Nanog may play a role in the process of adult cell segregation, but simple but simple. Overexpression of Nanog is not enough to dedifferentiate adult cells into totipotent cells.
We found that Nanog plays a vital role in the self-renewal process of embryonic stem cells. As a transcription factor, Nanog maintains the undifferentiated state of ES cells by promoting the expression of omnipotent related genes and inhibiting the expression of differentiation related genes, and the PI3K signaling pathway participates in the omnipotent of embryonic stem cells. Maintain, use LY294002 (PI3K pathway specific inhibitor) to inhibit the differentiation of ES cells after PI3K pathway. Exogenous overexpression of Nanog weakens the differentiation due to LY294002, but does not completely reverse this differentiation, which indicates that only part of the role of PI3K signaling pathway in ES cells is by regulating the expression of Nanog. To mediate, Nanog plays a role in the dedifferentiation of adult cells, but simple Nanog overexpression is not sufficient to dedifferentiate adult cells into an omnipotent stem cell stage.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R329

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