Foxa2在肝细胞饥饿状态下基因表达转录调控中的作用及机制

发布时间：2018-07-31 15:47

【摘要】：肝糖异生作用是长期饥饿下机体维持血糖平衡的主要途径,其主要依靠激素的调节,但不适当的调节可引起糖尿病和其他遗传代谢性疾病。糖异生途径中关键限速酶的调节是最基础和最基本的调节方式,其调控主要发生在转录水平,需要多种转录因子协调作用以适应机体不同的代谢和激素状态。在饥饿状态下,血胰岛素水平降低,胰高血糖素和糖皮质激素水平增高,分别通过活化 cAMP 应答元件结合蛋白(CREB)和糖皮质激素受体(GR)而促进糖异生作用。CREB 和 GR可以表达在多器官组织中,但是糖异生作用主要发生在肝脏,极少量发生在肾脏。“翼螺旋” 转录因子家族中的肝脏核转录因子 Foxa 的转录结合位点常与 CREB 和 GR 的结合位点相邻。但糖异生的活化过程中是否需要 Foxa 转录因子以及活化的分子机制尚未阐明。本课题建立了肝细胞转录因子 Foxa2 基因特异性剔除小鼠作为体内研究模型,并以从该小鼠分离培养得到的原代肝细胞为体外研究模型,采用免疫荧光染色技术(IFC)、免疫组织化学染色技术(IHC)、实时荧光定量 PCR 技术(RT-PCR)以及染色质免疫沉淀技术(CHIP assay),较系统、全面地对 Foxa2 在 cAMP 和糖皮质激素介导的肝细胞特异性基因 PEPCK、TAT和 IGFBP-1 转录活化中的作用和分子机理进行了研究,结果发现: 肝细胞 Foxa2 基因特异性剔除小鼠中 PEPCK、TAT 和 IGFBP-1 基因在饥饿状态下 mRNA 表达明显受抑;体外实验结果显示肝细胞中PEPCK、TAT 和 IGFBP-1 基因 mRNA 表达对不同的激素信号刺激,其
[Abstract]:Heterogenesis of liver sugar is the main way to maintain the balance of blood glucose under long-term starvation. It mainly depends on hormone regulation, but inappropriate regulation can cause diabetes mellitus and other genetic metabolic diseases. The regulation of key rate-limiting enzymes is the most basic and basic regulation in the glucose allogeneic pathway, which mainly occurs at the transcriptional level, and requires the coordination of various transcription factors to adapt to different metabolism and hormone status of the body. In hunger, blood insulin levels decreased, glucagon and glucocorticoid levels increased, CAMP response element binding protein (CREB) and glucocorticoid receptor (GR) were activated to promote the expression of glycosylated elements. CREB and gr could be expressed in multiple organ tissues respectively. However, glycosylation mainly occurred in the liver and a very small amount in the kidney. The transcriptional binding sites of liver nuclear transcription factor Foxa in the pterygoid transcription factor family are often adjacent to those of CREB and gr. However, whether Foxa transcription factors are needed in the process of glycosylation and the molecular mechanism of activation have not been elucidated. In this study, a mouse model of hepatocyte transcription factor Foxa2 gene specific knockout was established, and the primary hepatocytes isolated from the mouse were used as the in vitro study model. Immunofluorescence staining (IFC), immunohistochemical staining (IHC), real-time fluorescent quantitative PCR (RT-PCR) and chromatin immunoprecipitation (CHIP assay),) were used. The role and molecular mechanism of Foxa2 in the transcriptional activation of cAMP and glucocorticoid mediated hepatocyte specific gene PEPCK tat and IGFBP-1 were studied. The results showed that the mRNA expression of PEPCK tat and IGFBP-1 genes in hepatocyte Foxa2 gene specific knockout mice was significantly inhibited under starvation. The results of in vitro experiments showed that the expression of PEPCKT tat and IGFBP-1 gene mRNA in hepatocytes stimulated different hormone signals.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R33

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