DEPTOR的表达调控及功能研究

发布时间：2018-01-09 18:33

本文关键词：DEPTOR的表达调控及功能研究　出处：《华中农业大学》2016年博士论文　论文类型：学位论文

【摘要】：肝脏是人体最重要的器官之一,在代谢和免疫方面发挥着重要作用。肝脏通过肝门静脉接受来自肠道的静脉血,致使肝脏持续的暴露在病原体、毒素和食物抗原的环境下。在病理状态下,各种肝脏疾病往往伴随着肝脏炎症的发生,如脂肪肝和肝细胞癌。部分肝细胞癌患者肿瘤组织中DEPTOR的表达比邻近组织的高,尤其是伴随有B型肝炎或预后差的患者。DEPTOR是2009年新发现的一个mTOR抑制因子,参与细胞存活、增殖、成脂分化和自噬等多方面的调控。有研究报道DEPTOR与血管内皮细胞的激活及小鼠骨骼肌代谢性炎症相关。然而,DEPTOR是否参与肝脏炎症目前并未报道。因此,研究DEPTOR是否参与肝脏炎症及其表达调控与作用,不仅有助于了解机体的正常生理调控,也有助于揭示炎症相关疾病发生发展的分子机制并为治疗提供理论依据。本研究利用C57BL/6雌性小鼠、Hepa1-6细胞和HEK293T细胞,通过小鼠腹腔注射LPS构建炎症模型、细胞转染、实时定量PCR、免疫印迹、染色体免疫共沉淀、双荧光素酶报告系统、位点突变载体构建、ELISA、MitoTracker Green FM线粒体染色等方法,对DEPTOR在肝脏炎症中的表达调控及其作用进行了一系列研究,获得如下研究结果:1.构建了LPS诱导的小鼠炎症模型。与对照组相比,LPS诱导的小鼠肝脏组织和脂肪组织DEPTOR mRNA表达显著性下降,肌肉组织DEPTOR mRNA呈下降趋势但因个体差异较大下降未达到显著性水平。LPS诱导的小鼠肝脏组织DEPTOR蛋白降低,而mTORC1/2信号通路活性明显增加。2.在体外,利用小鼠肝细胞系(Hepa1-6细胞)验证了LPS能够下调DEPTOR的表达。3.LPS诱导的小鼠肝脏组织和Hepa1-6细胞中p65的表达上调。Hepa1-6细胞超表达p65下调DEPTOR mRNA和蛋白表达,且p65与LPS呈叠加效应下调DEPTOR蛋白表达。NF-κB抑制物IκBα和PDTC能够显著性削弱LPS对DEPTOR mRNA的下调。因此,LPS通过p65下调DEPTOR的表达。4.启动子转录因子结合位点预测到DEPTOR启动子区含有NF-κB结合位点。染色体免疫共沉淀分析、双荧光素酶报告系统和位点突变载体构建实验证实p65能够与DEPTOR启动子-145/-127区域结合,并下调DEPTOR启动子活性。5.Hepa1-6细胞超表达或者敲减DEPTOR促进巨噬细胞趋化因子MCP-1的表达。6.Hepa1-6细胞超表达DEPTOR促进细胞糖吸收、糖原积累和下调细胞线粒体数目。综上,LPS通过p65信号途径抑制DEPTOR的表达,而超表达DEPTOR则促进炎症因子MCP-1的表达。此外,DEPTOR还能够促进肝细胞糖吸收、糖原积累和下调肝细胞线粒体数目。
[Abstract]:The liver is one of the most important organs of the human body, plays an important role in metabolism and immunity. The liver via the hepatic portal vein receives venous blood from the intestines of the liver, resulting in continued exposure to pathogens, toxins and food antigens environment. In pathological conditions, various liver diseases are often accompanied by liver inflammation, such as fatty liver and liver cancer cells. DEPTOR expression of tumor tissue of patients with hepatocellular carcinoma adjacent tissues, especially with hepatitis B or poor prognosis of patients with.DEPTOR is a new discovery in 2009 mTOR inhibitory factor involved in cell survival, proliferation, differentiation and regulation of lipid into autophagy etc.. Studies have reported that DEPTOR and the activation of endothelial cells and mouse skeletal muscle metabolic inflammation. However, whether DEPTOR is involved in inflammation of the liver is not currently reported. Therefore, study on the involvement of DEPTOR in the liver Inflammation and regulation of its expression and function, not only helps to understand the normal physiological regulation of the body, but also helps to reveal the molecular mechanism of the occurrence and development of inflammation related diseases and provide theoretical basis for the treatment. This study used C57BL/6 female mice, Hepa1-6 cells and HEK293T cells, construct the inflammation model through the intraperitoneal injection of LPS transfection. Real time quantitative PCR, Western blotting, chromatin immunoprecipitation, dual luciferase reporter system, vector construction, mutation of ELISA, MitoTracker Green FM of mitochondrial staining, the expression of DEPTOR in liver inflammation and its role in the regulation of a series of research, the main results are as follows: 1. construct mice inflammatory model induced by LPS compared with the control group, the liver tissue of mice and adipose tissue DEPTOR mRNA expression induced by LPS significantly decreased, muscle tissue DEPTOR mRNA decreased due to a Large differences in body decline did not reach significant levels of.LPS induced mouse liver DEPTOR protein decreased, while mTORC1/2 increased the activity of.2. signaling pathway in vitro, using mouse liver cell line (Hepa1-6 cells) to verify the liver tissue of mice and Hepa1-6 cells expressing.3.LPS LPS can downregulate DEPTOR induced the expression of p65 was upregulated in.Hepa1-6 cells over expression of p65 and down-regulation of DEPTOR protein expression and mRNA, p65 and LPS showed a superimposed effect of down-regulation of DEPTOR protein expression of.NF- kappa B inhibitor I kappa B alpha and PDTC can significantly weaken the LPS of DEPTOR downregulation of mRNA. Therefore, LPS through downregulation of p65 binding site prediction to start DEPTOR promoter containing NF- B binding site sub the expression of.4. transcription factor DEPTOR. Analysis of chromosome immunoprecipitation, dual luciferase reporter system and mutation vector experiment demonstrated that p65 and DEPTOR promoter region -145/-127 Binding domain, and down-regulation of DEPTOR promoter activity in.5.Hepa1-6 cells overexpression or knockdown of DEPTOR promotes the over expression of DEPTOR promotes cell glucose uptake in cells expressing.6.Hepa1-6 MCP-1 gene more macrophages, glycogen accumulation and downregulation of cell number of mitochondria. In conclusion, LPS inhibits expression of DEPTOR through p65 signaling pathway, while overexpression of DEPTOR can promote the expression of MCP-1 in inflammatory factor. In addition, DEPTOR can also promote liver cell glucose uptake, glycogen accumulation and reduced number of liver mitochondria.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R575

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