【作者】 丁妙；

【导师】 谭拥军；

【作者基本信息】 湖南大学 ， 生物医学工程， 2010， 硕士

【摘要】 FoxA1(又名HNF-3α)是Fox翼状螺旋转录因子家族成员之一。在发育的神经管以及成体的脑组织中FoxA1的表达模式表明其在神经形成起到了重要作用。为了进一步阐明转录因子FoxA1诱导多能性干细胞分化的基因调控机理,本文主要探索干细胞神经细胞分化过程中,转录因子FoxA1对Nestin基因表达的调节作用。我们采用小鼠胚胎癌细胞-维甲酸诱导分化的细胞模型,发现分化发生的最早期,FoxA1表达上调,随后神经发育基因Nestin的表达随神经干细胞的分化上调,推测Nestin可能受FoxA1的直接调控。为此,我们对神经发育过程中FoxA1对Nestin的表达调节作进一步的研究。采用高表达FoxA1转录因子和干扰FoxA1转录因子两种手段来考察Nestin的表达对应关系。进一步采用染色质免疫共沉淀的方法分析FoxA1的表达水平与FoxA1蛋白是否能够能源性结合到Nestin的启动子区域。构建了Nestin的启动子介导的报告基因质粒,通过共转染实验考察FoxA1对Nestin启动子的转录刺激作用。转录因子FoxA1在胚胎发育时期扮演了重要的角色,该因子在维甲酸诱导多能性畸胎癌细胞神经分化早期被激活,同时增加Nestin的表达水平。在多能性畸胎癌P19干细胞内单独过表达FoxA1可以刺激Nestin的表达。同时,当在P19细胞神经分化过程中抑制FoxA1的表达时,导致Nestin的表达阻断。FoxA1通过结合到Nestin的-4179至-3919bp的启动子区域启动该基因的表达。在P19细胞内通过腺病毒介导的高表达Foxa1实验,证明Nestin的表达与FoxA1存在相关性。在维甲酸诱导P19细胞神经过程中,增加FoxA1的表达水平可以增加神经元样细胞的数量。总之,本研究证明FoxA1在促进多能性干细胞的神经分化早期,起到了重要的作用。更多还原

【Abstract】 Transcription factor FoxA1 (previously known as HNF-3α) is belongs to the fork head/winged-helix family of transcription factors.The expression pattern of FoxA1 in developing neural tube and adult brain structures implicates its important roles in neurogenesis.To further clarify the gene regulatory mechanism of the transcription factor Foxa1 induced differentiation of the embryonic stem cell, this paper is to study the regulate role of Transcription Foxal to the Nestin expression in the neural differentiation stage of the stem cell.By using the mode1 of RA-induced P19 cell neural differentiation, we discovered that Foxal was expressed at a very high levelaccompanied by the decline of nanog, and the expresseion of neural development gene Nestin was up-regulated with the polarization of a stem cell.Therefore, we further studied the regulation of gene Nestin in the neural development.Furthermore, we investigated the change of Nestin by overexpressing and Knocking-down FoxAl respectively by foxa1 siRNA.The CHIP experiment was further conducted to verify that whether Foxa1 protein can bind to the promoter of Nestin, meanwhile, the reporter gene plasmid was constructed through the promoter of Nestin, together with the co-transfection experiment to examine the role of FoxA1 to Nestin.Transcription factor FoxAl plays a critical role during embryonic development and is activated during retinoic acid (RA)-induced neural differentiation of pluripotent P19 embryonal carcinoma cells at the early stage, which is marked by increased expression of neural stem cell marker Nestin.we have overexpressed FoxAl through an adenovirus vector in P19 cells and identified that early neurogenesis-related Nestin gene is activated directly by FoxAl.Furthermore, Knockdown of FoxAl expression during P19 cell neural differentiation results in prevention of Nestin induction. FoxAl binds to Nestin promoter at-4179 to-3919 bp region and activates the promoter in cotransfection assays.During RA-induced P19 cell differentiation, elevated levels of FoxAl increase the population of neurons.Together, our results suggest a critical involvement of FoxAl in stimulating neural differentiation of pluripotent stem cells at early stages.更多还原