本文關(guān)鍵詞：DEPTOR的表達(dá)調(diào)控及功能研究　出處：《華中農(nóng)業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： DEPTOR 炎癥 p65 脂多糖 mTOR 轉(zhuǎn)錄調(diào)控 糖吸收 線粒體

【摘要】：肝臟是人體最重要的器官之一,在代謝和免疫方面發(fā)揮著重要作用。肝臟通過肝門靜脈接受來自腸道的靜脈血,致使肝臟持續(xù)的暴露在病原體、毒素和食物抗原的環(huán)境下。在病理狀態(tài)下,各種肝臟疾病往往伴隨著肝臟炎癥的發(fā)生,如脂肪肝和肝細(xì)胞癌。部分肝細(xì)胞癌患者腫瘤組織中DEPTOR的表達(dá)比鄰近組織的高,尤其是伴隨有B型肝炎或預(yù)后差的患者。DEPTOR是2009年新發(fā)現(xiàn)的一個mTOR抑制因子,參與細(xì)胞存活、增殖、成脂分化和自噬等多方面的調(diào)控。有研究報道DEPTOR與血管內(nèi)皮細(xì)胞的激活及小鼠骨骼肌代謝性炎癥相關(guān)。然而,DEPTOR是否參與肝臟炎癥目前并未報道。因此,研究DEPTOR是否參與肝臟炎癥及其表達(dá)調(diào)控與作用,不僅有助于了解機(jī)體的正常生理調(diào)控,也有助于揭示炎癥相關(guān)疾病發(fā)生發(fā)展的分子機(jī)制并為治療提供理論依據(jù)。本研究利用C57BL/6雌性小鼠、Hepa1-6細(xì)胞和HEK293T細(xì)胞,通過小鼠腹腔注射LPS構(gòu)建炎癥模型、細(xì)胞轉(zhuǎn)染、實時定量PCR、免疫印跡、染色體免疫共沉淀、雙熒光素酶報告系統(tǒng)、位點(diǎn)突變載體構(gòu)建、ELISA、MitoTracker Green FM線粒體染色等方法,對DEPTOR在肝臟炎癥中的表達(dá)調(diào)控及其作用進(jìn)行了一系列研究,獲得如下研究結(jié)果:1.構(gòu)建了LPS誘導(dǎo)的小鼠炎癥模型。與對照組相比,LPS誘導(dǎo)的小鼠肝臟組織和脂肪組織DEPTOR mRNA表達(dá)顯著性下降,肌肉組織DEPTOR mRNA呈下降趨勢但因個體差異較大下降未達(dá)到顯著性水平。LPS誘導(dǎo)的小鼠肝臟組織DEPTOR蛋白降低,而mTORC1/2信號通路活性明顯增加。2.在體外,利用小鼠肝細(xì)胞系(Hepa1-6細(xì)胞)驗證了LPS能夠下調(diào)DEPTOR的表達(dá)。3.LPS誘導(dǎo)的小鼠肝臟組織和Hepa1-6細(xì)胞中p65的表達(dá)上調(diào)。Hepa1-6細(xì)胞超表達(dá)p65下調(diào)DEPTOR mRNA和蛋白表達(dá),且p65與LPS呈疊加效應(yīng)下調(diào)DEPTOR蛋白表達(dá)。NF-κB抑制物IκBα和PDTC能夠顯著性削弱LPS對DEPTOR mRNA的下調(diào)。因此,LPS通過p65下調(diào)DEPTOR的表達(dá)。4.啟動子轉(zhuǎn)錄因子結(jié)合位點(diǎn)預(yù)測到DEPTOR啟動子區(qū)含有NF-κB結(jié)合位點(diǎn)。染色體免疫共沉淀分析、雙熒光素酶報告系統(tǒng)和位點(diǎn)突變載體構(gòu)建實驗證實p65能夠與DEPTOR啟動子-145/-127區(qū)域結(jié)合,并下調(diào)DEPTOR啟動子活性。5.Hepa1-6細(xì)胞超表達(dá)或者敲減DEPTOR促進(jìn)巨噬細(xì)胞趨化因子MCP-1的表達(dá)。6.Hepa1-6細(xì)胞超表達(dá)DEPTOR促進(jìn)細(xì)胞糖吸收、糖原積累和下調(diào)細(xì)胞線粒體數(shù)目。綜上,LPS通過p65信號途徑抑制DEPTOR的表達(dá),而超表達(dá)DEPTOR則促進(jìn)炎癥因子MCP-1的表達(dá)。此外,DEPTOR還能夠促進(jìn)肝細(xì)胞糖吸收、糖原積累和下調(diào)肝細(xì)胞線粒體數(shù)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.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R575

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