Nanog在胚胎干细胞自我更新中的作用机制研究

【作者】 刘娜；

【导师】 韩忠朝； 陆敏；

【作者基本信息】 中国协和医科大学 ， 内科学， 2008， 博士

【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。更多还原

【Abstract】 Embryonic stem cells (ES cells) have two defining properties, self-renewal and pluripotency, which makes them very attractive in clinic. Fully understanding of the mechanism involved in the pluripotency and early differentiation is essential for achieving these goals. Nanog, a homeodomain-containing transcription factor, was found at 2003 and specially expressed in pluripotency cells. Nanog plays important roles in ES cells pluripotency, early differentiation and somatic cells dedifferentiation. But its mechanism is not well elucidated. Here, we aimed to illustrate the mechanism of Nanog in embryonic stem cells by studying the target genes, the related signaling pathway and the effect in the somatic cells dedifferentiation.The purpose of the first part of this paper is to investigate the target genes which be regulated by Nanog. Nanog, as a transcription factor, produces effects by regulating target genes expression. The information about these target genes is helpful for us to well known the mechanism of Nanog in embryonic stem cells self-renewal. Here, we aim to investigate the genes possibly participating in the course of self-renewal regulated by Nanog by combining RNA interference and microarray detection method. In order to down-regulate Nanog expression efficiently, four siRNAs were designed on the basis of the conserved Nanog sequence and their effects on the Nanog expression were tested by Western-Blot and Real-Time PCR. Among these four siRNAs, Nanog-siRNA-P1 was found to be most effective. The interference efficience was up to 90%. Once Nanog was down-regulated, ES cells underwent differentiation by showing morphological change and decreased proliferation rate. Microarray analysis was then used to identify the altered gene expression at 24 or 48 hours after Nanog was silenced. These changed genes in our experiment were considered as the result of Nanog downregulation, and this would help us to known better the mechanism of Nanog in the pluripotency of ES cells. In the second part, we investigated the effect of PI3K signaling transduction pathway on ES cells self-renewal and Nanog expression. We found that LY294002, a specific inhitor of PI3K pathway, induced mouse ES cells differentiation and decreased Nanog expression. To investigate the mechanism of PI3K signaling on ES cells self-renewal, we constructed a Nanog-GFP fusion protein expression plasmid, and then trasfected ES cells. After G418 selection, we got Nanog-overexpressing ES cells (Ex-Nanog-J1). Exogenous Nanog sustained mouse ES cells pluripotency independent of LIF, and alleviates the differentiation induced by LY294002. But it was insufficient to totally reverse the differentiation.In the third part, we tried to investigate the effect of Nanog in somatic cells dedifferentiation (reprogramming). We all know that ES cells have the ability to differentiate into all kind cells and tissues of three germinal layers. And, somatic cells can be dedifferentiated to stem cells by cell fusion and somatic cells nuclear transfer (SCNT). Nanog is expressed specifically in embryonic stem cells, and plays an important role in embryonic stem cells self-renewal. Then, we want to know the effect of Nanog in somatic cells dedifferentiation. To investigate the effect of Nanog in somatic cells dedifferentiation, we constructed Nanog-GFP fusion protein expression vector and then transfected it to 3T3 cells. After G418 selection, we got Nanog-expressing 3T3 cells. We observed that Nanog was located in 3T3 cells nuclear, which was similar with the location of Nanog in embryonic stem cells. Then we investigated if the 3T3 cells with Nanog-overexpression could dedifferentiate. There was no significant differentiation in the cells morphology. Cell cycle results showed that S phase was increased and G0/G1 phase and M phase were decreased in 3T3 cells with Nanog expression compared with wild-type 3T3 cells. Furthermore, we observed that Oct4 expressed in 3T3 cells with exogenous Nanog. But, other ES cells specific genes, such as Sox2, Utf1, and Ets2 were not detected in 3T3 cells with Nanog overexpression. All of these evidences indicated that Nanog might take part in the somatic cells dedifferentiation. But only exogenous Nanog was insufficient to promote somatic cells dedifferentiated totally.This investigation shows that Nanog plays a key role in ES cells self-renewal. Nanog, as a transcription factor, sustains ES cells undifferentiated state by activating pluripotency related genes expression and repressing differentiation related genes expression. PI3K signaling pathway plays a significant role in embryonic stem cells self-renewal. LY294002, a specific inhibitor of PI3K signaling, induced ES cells differentiation. Exogenous Nanog alleviates the differentiation induced by LY294002. But it’s insufficient to totally reverse the differentiation. So, we think that the PI3K-dependent regulation of ES cells self-renewal is partially mediated by regulated Nanog expression. Nanog is helpful for somatic cells dedifferentiation but only Nanog overexpression was insufficient to induced somatic cells to pluripotency cells.更多还原