本文選題：NDRG2　切入點：脂肪酸氧化　出處：《第四軍醫(yī)大學(xué)》2017年博士論文

【摘要】：在腫瘤的形成和發(fā)展過程中,常常伴隨著營養(yǎng)物質(zhì)和氧氣的供給不足[1]。特別是在實體瘤的迅速增長過程中,由于相對血供不足,常導(dǎo)致瘤體內(nèi)的許多區(qū)域缺乏氧氣和葡萄糖等營養(yǎng)物質(zhì)[2]。在這種情況下,腫瘤細胞必須通過代謝模式的改變以適應(yīng)惡劣的微環(huán)境。腫瘤細胞代謝重編程最為熟知的當(dāng)屬Warburg效應(yīng),其典型特征是主要通過增強產(chǎn)能效率低下的糖酵解途徑來生產(chǎn)ATP并伴隨大量乳酸的形成[3]。除此之外,腫瘤細胞也常常發(fā)生谷氨酰胺代謝和脂代謝的重編程[4],例如在缺乏葡萄糖等應(yīng)激情況下,腫瘤細胞通過增強脂肪酸氧化分解來提供能量和營養(yǎng)物質(zhì),維持細胞存活[5,6]。由于這種對代謝應(yīng)激的適應(yīng)能力,在臨床抗血管生成藥物治療后,復(fù)發(fā)的腫瘤細胞甚至表現(xiàn)出更強的增殖和轉(zhuǎn)移能力[7]。因此,研究腫瘤代謝重編程的分子機制具有重要的理論意義,也能為腫瘤的分子靶向治療提供新策略。NDRG2是我們課題組首先報道的抑癌基因,在人體多種腫瘤組織中低表達,過表達NDRG2后可以抑制多種腫瘤細胞系的增殖、遷移和侵襲[8]。另外,NDRG2還參與細胞的多種應(yīng)激反應(yīng)[9]。更為重要的是,越來越多的研究證實NDRG2與腫瘤細胞代謝有密切關(guān)系。我們前期的研究發(fā)現(xiàn),NDRG2能夠抑制乳腺癌細胞的葡萄糖攝取和結(jié)腸癌細胞的糖酵解和谷氨酰胺代謝[10,11]。然而NDRG2是否參與腫瘤細胞脂代謝還未見報道�；诖�,本課題擬以過表達NDRG2的肝癌細胞為研究模型,探討實體瘤因微環(huán)境異質(zhì)性和基質(zhì)脫落等代謝應(yīng)激條件下NDRG2在肝癌細胞中的作用。目的:1.明確代謝應(yīng)激時NDRG2對肝癌細胞存亡的影響2.研究缺糖應(yīng)激時NDRG2對肝癌細胞能量及氧化還原狀態(tài)的影響3.探究NDRG2在肝癌細胞缺糖應(yīng)激中所起作用的分子機制方法:1.利用慢病毒感染構(gòu)建過表達NDRG2的肝癌細胞系Huh7和Sk-hep-1及表達Cherry的對照細胞,q PCR和Western blot檢測NDRG2的表達情況;CCK8實驗檢測和比較上述細胞在正常培養(yǎng)和代謝應(yīng)激情況下的細胞活力;克隆形成實驗檢測正常培養(yǎng)和缺糖應(yīng)激下的克隆形成能力;流式細胞術(shù)和TUNEL染色檢測細胞凋亡。2.分別用ATP檢測試劑盒、NADP/NADPH定量比色試劑盒和DCFH-DA探針檢測NDRG2過表達和對照肝癌細胞在正常培養(yǎng)和缺糖應(yīng)激情況下的細胞內(nèi)ATP水平、NADPH含量和NADP+/NADPH比值以及細胞內(nèi)ROS水平。3.q PCR檢測過表達NDRG2和對照肝癌細胞在正常培養(yǎng)和缺糖應(yīng)激下脂肪酸氧化(FAO)關(guān)鍵調(diào)控分子的表達情況。給予PPARα的激活劑BEZA(400μM)和CPT1A的抑制劑ETO(100μM)后,分別用ATP檢測試劑盒、NADP/NADPH定量比色試劑盒和CCK-8檢測上述細胞在低糖應(yīng)激下的細胞內(nèi)ATP水平、NADPH含量以及細胞存活情況。4.Western blot檢測NDRG2過表達肝癌細胞在正常和缺糖情況下AMPK/ACC通路的活化情況(p-AMPKα,AMPKα,p-ACC和ACC);在NDRG2過表達細胞Huh7中強行表達組成性活化的AMPKα,Western blot比較AMPK信號通路的活化情況,流式細胞術(shù)比較細胞凋亡。結(jié)果:1.CCK-8實驗、克隆形成實驗和凋亡檢測結(jié)果表明,缺糖應(yīng)激條件下過表達NDRG2抑制肝癌細胞存活,降低肝癌細胞的克隆形成能力,促進缺糖應(yīng)激中肝癌細胞的凋亡。2.細胞內(nèi)ATP、NADPH和ROS檢測結(jié)果表明,過表達NDRG2降低缺糖應(yīng)激條件下肝癌細胞內(nèi)ATP和NADPH含量,升高NADP+/NADPH比值和細胞內(nèi)ROS水平,過表達NDRG2加劇了缺糖應(yīng)激下肝癌細胞能量和氧化還原失衡。3.NDRG2過表達可抑制缺糖應(yīng)激誘導(dǎo)的脂肪酸氧化相關(guān)分子PPARα、CPT1A和ACADM的表達上調(diào)及脂肪酸氧化的活化。PPARα的激動劑BEZA可增加缺糖應(yīng)激時NDRG2過表達肝癌細胞內(nèi)ATP和NADPH含量,從而削弱缺糖時NDRG2對細胞存活的抑制作用。反之,CPT1A的抑制劑ETO則顯著降低細胞ATP和NADPH含量,抑制缺糖應(yīng)激下的細胞存活,該作用在對照細胞中比NDRG2過表達細胞更明顯。4.NDRG2過表達可抑制缺糖應(yīng)激條件下肝癌細胞中AMPK信號通路的活化;組成性活化的AMPK可逆轉(zhuǎn)NDRG2對AMPK信號通路的抑制作用并部分緩解缺糖應(yīng)激誘導(dǎo)的肝癌細胞凋亡。結(jié)論:缺糖應(yīng)激條件下過表達NDRG2通過抑制肝癌細胞AMPK信號通路的活化,干擾脂肪酸氧化的激活,加劇肝癌細胞的能量耗竭和氧化應(yīng)激并導(dǎo)致細胞凋亡,最終降低缺糖應(yīng)激時肝癌細胞的存活能力。
[Abstract]:In the process of formation and development of tumor, often accompanied by the insufficient supply of nutrients and oxygen to [1]. especially in the rapid growth of solid tumors in the process, due to the relatively insufficient blood supply, often leads to many areas of tumors lack of oxygen and glucose, nutrients such as [2]. in this case, tumor cells must through metabolic mode change in order to adapt to the harsh environment. The effect of Warburg micro is known as tumor cell metabolic reprogramming, its typical character is mainly by enhancing the capacity of low efficiency glycolysis to produce ATP and [3]. formed in addition with large amounts of lactic acid, tumor cells often occur and lipid metabolism of glutamine reprogramming [4], for example, in the absence of glucose stress tumor cells by enhanced fatty acid oxidation to provide energy and nutrients to maintain cell survival by [5,6]. in the The metabolic stress adaptation in clinical anti angiogenesis drugs after treatment, the recurrence of tumor cells showed even stronger proliferation and metastasis of [7]., therefore, has an important theoretical study on the molecular mechanism of tumor metabolic reprogramming, but also for tumor molecular targeted therapy provides a new strategy for.NDRG2 is an oncogene and our group first reported, low expression in a variety of human tumor tissues, overexpression of NDRG2 can inhibit tumor cell proliferation, migration and invasion of [8]. and NDRG2 were involved in various stress responses [9]. is even more important, more and more studies have confirmed that NDRG2 has a close relationship with the metabolism of tumor cells our previous study showed that NDRG2 can inhibit breast cancer cell glucose uptake in human colorectal cancer cells and glycolysis and glutamine metabolism in [10,11]. but NDRG2 is involved in tumor Cell lipid metabolism has not been reported. Based on this, this paper intends to over expression of NDRG2 in hepatocellular carcinoma cells as a model, to investigate the effect of solid tumors due to micro environmental heterogeneity and matrix off metabolic stress under the condition of NDRG2 in hepatocellular carcinoma. Objective: 1. clear metabolic stress effects of NDRG2 on liver cancer cell survival of 2. glucose deprivation stress NDRG2 redox state effect on hepatoma cells and explore the 3. energy oxidation of NDRG2 in hepatoma cell glucosedeprivation plays stress molecular mechanism of the effect of 1. methods: using lentiviral infection constructed over expression of NDRG2 in hepatocellular carcinoma cell line Huh7 and Sk-hep-1 and Cherry expression in control cells, the expression of Q PCR and Western blot detection NDRG2; cell viability by CCK8 assay and compared the cells in normal culture and metabolic stress conditions; colony forming assay and cloning of cultured normal glucose deprivation stress formation Force; flow cytometry and TUNEL staining to detect the apoptosis of.2. cells were detected by ATP kit, NADP/NADPH quantitative colorimetric kit and DCFH-DA probe for detection of NDRG2 expression and control of hepatoma cells in normal culture and lack of sugar should be ATP level below passionate condition in cells, NADPH content and NADP +/NADPH ratio and intracellular ROS level.3.q PCR tested the expression of NDRG2 and control cells cultured under normal glucose deprivation stress and fatty acid oxidation (FAO) expression of key regulatory molecules. PPAR alpha activator BEZA (400 M) and CPT1A inhibitor ETO (100 M), were detected by ATP kit, NADP/NADPH quantitative colorimetric detection kit and CCK-8 cells in the low glucose stress under the intracellular ATP level, NADPH content and cell survival of.4.Western blot detection NDRG2 hepatocellular carcinoma cells in normal and low glucose conditions AMPK/ACC pathway activity Situation (p-AMPK alpha, alpha AMPK, p-ACC and ACC); in NDRG2 overexpressing Huh7 cells expressing a constitutively activated AMPK by activation of Western blot alpha, AMPK signaling pathway, flow cytometric analysis of cell apoptosis. Results: the 1.CCK-8 assay, clone formation assay and apoptosis detection results show that the lack of sugar stress overexpression of NDRG2 inhibits the survival of cancer cells, cancer cells reduced the colony forming ability, promote the lack of sugar stress in hepatocellular carcinoma cell apoptosis of.2. cells in ATP, NADPH and ROS results showed that overexpression of NDRG2 reduced glucose deprivation stress conditions in hepatocellular carcinoma cell line ATP and the content of NADPH, increase the level of ROS NADP+/NADPH ratio and in cells, overexpression of NDRG2 increased cell energy and oxidation of glucose deprivation stress reduction imbalance.3.NDRG2 overexpression inhibits glucose deprivation stress induced fatty acid oxidation related molecule PPAR alpha, CPT1A expression and ACADM. And fatty acid oxidation of activated.PPAR alpha agonist BEZA could increase glucose deprivation stress NDRG2 expression content of ATP and NADPH cells, thus weakening the lack of sugar when the inhibitory effect of NDRG2 on cell survival. On the contrary, the CPT1A inhibitor ETO significantly decreased the content of NADPH and ATP cells, inhibit glucose deprivation stress the role of cell survival in control cells than in NDRG2 overexpressing cells more obvious overexpression of.4.NDRG2 can inhibit glucose deprivation stress activation of AMPK signaling pathway in hepatocellular carcinoma cells; constitutively activated AMPK can reverse the NDRG2 of the AMPK signaling pathway inhibition and apoptosis of hepatocellular carcinoma cells with partial remission induced by glucose deprivation stress. Conclusion glucose deprivation stress conditions: overexpression of NDRG2 by inhibiting the activation of AMPK signaling in hepatocellular carcinoma cells, activate the interference of fatty acid oxidation, increased energy depletion and oxidative stress in liver cancer cells and induce cell apoptosis, The survival ability of hepatoma cells was reduced in the end.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R735.7
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本文編號：1697160