【作者】 牟玉莲；

【导师】 李奎；

【作者基本信息】 中国农业科学院 ， 动物遗传育种与繁殖， 2009， 博士

【摘要】 胚胎干细胞是从囊胚内细胞团分离出来的,具有无限增殖、自我更新的能力。其全能性分子机制的研究是多年来的研究热点。Tle4基因是长型Groucho/Tle基因家族的成员之一,包括Q区、WD40区、GP区、CcN基序和SP区5个部分。小鼠Tle4基因位于19号染色体,全长约为234.45kb,包含20个外显子和19个内含子,编码773个氨基酸。差减RNA干扰文库筛选结果提示其是维持ES细胞多能性的靶基因。本论文对Tle4基因的转录调控及其在ES细胞功能进行了相对系统的研究,为深入研究和理解ES细胞多能性维持的分子机制提供科学依据,具有重要的意义。研究内容和结果如下:(1)首先利用生物信息学对小鼠Tle4基因进行了分析,发现其编码的蛋白含有7个重复WD40结构域,并且在物种间高度保守。同时通过数据库芯片表达谱分析,表明其在癌细胞、免疫细胞、胚胎干细胞表达量均很高,可能与细胞分化、增殖相关。(2)利用生物信息学对小鼠Tle4基因5’侧翼序列特征进行了分析,并对Tle4基因5’侧翼区域进行克隆、鉴定与初步分析。采用PCR定向克隆和酶切亚克隆策略,构建了覆盖Tle4基因5’侧翼起始密码子ATG上游2.8kb区域的7个不同片段长度的缺失报告基因重组载体,用promega的双荧光素酶报告实验系统分别在小鼠ES细胞、畸胎瘤(F9)细胞中检测其活性。结果显示,Tle4基因核心调控区域定位于-2137bp～-1794bp区域(翻译起始密码子ATG中的A定为+1)。利用转录因子结合位点预测软件分析表明,此区域有比较密集的潜在转录因子结合位点,可能含有重要的转录激活元件,为小鼠Tle4基因的关键启动区。另外,还提示在-2521bp～-2137bp区域含有负调控元件。(3)对-2137bp～-1794bp区域再次进行缺失,构建了3个缺失片段-pGL3重组质粒。结果发现-2027bp～-1927bp区域荧光素酶活性最强,表明其是Tle4基因的核心调控区域。生物信息学分析发现,此区域存在Tcf1、Lef1、Sp1等多个转录因子的结合位点,它们可能参与Tle4基因的转录调控。(4)进行Tle4基因核心调控区域的定点突变分析。针对已经初步鉴定的Tle4基因核心调控区域Tcf1结合位点序列,构建Tle4基因荧光素酶报告基因定点突变载体。活性分析表明,Tcf1结合的保守位点突变后,其活性有所降低,但差异不显著,提示Tcf1可能在Tle4基因的转录调控中起着一定的作用,但不是必需的。(5)检测了Tle4基因在小鼠囊胚、胎儿和2d和6d EB的表达情况,结果显示其均表达。另外其在MEF中不表达。(6)根据Tle4基因序列设计合成了三对siRNA片段和一对无意义siRNA片段。利用Lipofectamine 2000脂质体将siRNA转染ES细胞系,转染培养后24h,利用总RNA提取试剂盒提取这些细胞总RNA,并经过半定量RT-PCR分析,筛选到干扰效果最好的一对siRNA。之后,将其转染ES细胞,发现干扰后的ES细胞在形态、碱性磷酸酶活性和分化能力等方面并没有发生变化,仍维持全能性的能力。(7)克隆了小鼠Tle4基因全长mRNA序列,构建了Tle4基因过表达载体,发现当Tle4基因过表达后,ES细胞发生了分离解体现象。同时构建了Tle4基因荧光融合蛋白表达载体,将Tle4蛋白定位于ES细胞的细胞核上。综上所述,本论文对小鼠Tle4基因转录调控及其在ES细胞的分子功能的系统研究,进一步加深了我们对Tle4分子功能和机制的认识,为深入开展Tle4基因的研究具有积极的理论和现实意义。更多还原

【Abstract】 Mouse embryonic stem (ES) cells are pluripotent cell lines isolated from the inner cell mass of blastulas which can proliferate unlimitedly and have ability of self-renewal. It is a hot spot for studying the molecular mechanisms of its developmental pluripotency. Tle4 gene is a member of Groucho/Tle gene family which includes five domains: the highly conserved glutamine (Q) rich, the WD-repeat domain, GP domain, CcN motif and SP domain. Mouse Tle4 gene is located in chromosome 19 that its full length is 234.45kb with 20 exons and 19 introns. It encodes 773 amino acid. The result from RNA interference library indicates Tle4 gene is a target gene that can maintain ES cells pluripotency. In the present study, we have systematically investigated the transcriptional regulation of Tle4 gene and its role in ES cells. The contents and results were summarized as followings:(1)At first, It has found that Tle4 protein contain seven conserved WD40 domains by bioinformatic analysis. At the same time, it was high expressed in tumor cells, immunity cells and ES cells, which indicates that it was related to cell differentiation and proliferation.(2)Characterization and identification of 5’-flanking region of Tle4 gene was analyzed by bioinformatic information. Seven overlapping genomic fragments from the 5’-flanking region of Tle4 gene were coloned into pGL3-basic vector to construct Tle4 promoter reporters. The transcriptional activities of these promoter fragments were tested in ES cells, F9 cells by dual luciferase assay. The results of software suggested that the -2137bp～-1794bp region (the first A nueleotide of the translation initiation codon ATG was numbered as+l) was the critical region, which contains many putative transcription factor binding sites and important element for transcriptional activation. In addition, the -2521bp～-2137bp region may contain negative regulatory elements.(3)Three different fragments of -2137bp～-1794bp region have been coloned into pGL3-basic vector for further analysis. The results suggested that transcriptional activity of -2027bp～-1927bp region was strongest and it was the core regulation region. Many transcription factor binding sites, such as Tcf1, Lef1, Sp1, et al, were found, which may be involved in the transcriptional control of Tle4 gene.(4) Analysis of site-directed mutations in Tle4 gene core regulation region. The site-directed mutations of Tcf1 fragment was coloned into pGL3-basic vector to detect. The transcriptional activity of mutation Tcf1-binding sites was repressed to some extent, but not significantly. This indicated that Tcf1 may play certain roles, but was not necessary.(5)Tle4 gene was expressed in blastulas, fetus, EB of 2d and 6d. It was not expressed in MEF.(6) According to the sequence of Tle4 gene, three pairs of siRNA fragments and one unspecific siRNA fragment were synthesized. These siRNA fragments were transfected with Lipofectamine 2000 into mouse ES cells. Total RNA was isolated from ES cells after 24 hours transfection by total RNA kit. The high efficient siRNA that significantly down-regulated Tle4 gene expression was selected by RT-PCR analysis. ES cells were transfected by Lipofectamine 2000 with the selected siRNA fragments. But the morphous, alkaline phosphatase activity, differentiation activity of ES cells were not changed and ES cells still maintained pluripotency after transfection.(7) The full-length mRNA sequence of Tle4 gene was cloned into vector for over-expression. ES cells were disintegrated after transfection with over-expression vector. The vector for subcelluar localization pEGFP-N1-Tle4 was also construed and Tle4 protein was localized to cell nucleus in ES cells.In summary, the study on transcriptional control and the role in ES cells of Tle4 gene can contribute to the understanding of the molecular function and mechanism of this gene. It has active theory and actual significance.更多还原