本文選題：營養(yǎng)缺乏環(huán)境 + 線粒體分裂/融合　； 參考：《第四軍醫(yī)大學》2016年博士論文

【摘要】：背景:線粒體是調(diào)控細胞代謝最重要的細胞器,通過不斷地分裂/融合調(diào)控自身功能并對外界刺激做出適應性反應。大量研究證實,線粒體分裂/融合異常與多種神經(jīng)退行性疾病、心血管疾病及代謝性疾病的發(fā)生進展密切相關(guān)。近年來研究證實,多種類型腫瘤中線粒體分裂/融合發(fā)生異常,并參與腫瘤惡性進展。實體瘤常由于生長過快而導致內(nèi)部出現(xiàn)慢性營養(yǎng)缺乏微環(huán)境,腫瘤細胞需感知并通過代謝重編程做出適應才能繼續(xù)生存,而目前腫瘤適應營養(yǎng)缺乏的機制仍不十分清楚,闡明其機制對腫瘤防治具有重大意義。我們前期研究發(fā)現(xiàn),營養(yǎng)缺乏環(huán)境時肝癌細胞線粒融合變長。作為細胞能量代謝調(diào)控核心的線粒體,其形態(tài)結(jié)構(gòu)的變化是否參與肝癌細胞在營養(yǎng)缺乏時的代謝適應尚不清楚。目的:1.明確營養(yǎng)缺乏環(huán)境對肝癌細胞線粒體分裂/融合的調(diào)控作用與機制。2.探討線粒體融合對肝癌細胞能量代謝的調(diào)控作用。3.分析線粒體融合調(diào)控能量代謝的腫瘤生物學意義。方法:1.利用mito-tracker熒光染色與透射電鏡(tem)技術(shù),在肝癌細胞系與肝癌臨床組織標本中分析營養(yǎng)缺乏環(huán)境對線粒體分裂與融合的調(diào)控作用。2.利用qrt-pcr與westernblot實驗,檢測營養(yǎng)缺乏環(huán)境下參與線粒體分裂/融合調(diào)控的關(guān)鍵分子及其上游信號分子的表達與活性,分析營養(yǎng)缺乏環(huán)境促進線粒體融合的分子機制。3.利用qrt-pcr、westernblot與gc-ms(氣相色譜-質(zhì)譜聯(lián)用)技術(shù),分析營養(yǎng)缺乏時線粒體融合對肝癌細胞糖酵解與氧化磷酸的調(diào)控。4.利用tem(透射電鏡)、bluenative-page、co-ip、qrt-pcr與westernblot技術(shù),對營養(yǎng)缺乏時線粒體融合調(diào)控氧化磷酸化與糖酵解的機制進行研究。5.利用fcm(流式細胞術(shù))與克隆形成實驗,分析營養(yǎng)缺乏時線粒體融合對肝癌細胞存活的影響。6.利用裸鼠皮下成瘤實驗,在體內(nèi)分析線粒體融合對腫瘤生長的影響。7.利用ihc實驗,對促進營養(yǎng)缺乏環(huán)境下線粒體融合關(guān)鍵分子的表達進行檢測,并對其與肝癌患者臨床參數(shù)及預后進行相關(guān)性分析。結(jié)果:1.mito-tracker熒光染色證實營養(yǎng)缺乏促進肝癌細胞線粒體融合變長;透射電鏡(tem)對肝癌組織線粒體形態(tài)觀察發(fā)現(xiàn),腫瘤中心區(qū)(營養(yǎng)缺乏)較邊緣區(qū)(營養(yǎng)充足)線粒體變長。2.營養(yǎng)缺乏時,線粒體分裂/融合調(diào)控關(guān)鍵分子drp1、fis1、mfn1、mfn2與opa1表達均未發(fā)生顯著變化,而線粒體定位的drp1因被pka磷酸化(s637)而減少,導致線粒體變長。3.營養(yǎng)缺乏時,線粒體融合促進肝癌細胞氧化磷酸而抑制糖酵解。4.營養(yǎng)缺乏時,線粒體融合通過使線粒體嵴緊密促進氧化呼吸鏈復合體組裝,從而激活氧化磷酸化而抑制糖酵解;線粒體融合通過氧化磷酸化介導的nad+/sirt1/hif-1α信號抑制糖酵解。5.線粒體融合抑制肝癌細胞在營養(yǎng)缺乏環(huán)境下的凋亡,促進肝癌細胞克隆形成能力。6.營養(yǎng)缺乏誘導的線粒體融合促進腫瘤生長。7.介導營養(yǎng)缺乏環(huán)境下線粒體融合的關(guān)鍵分子p-drp1s937表達水平與肝癌患者腫瘤分級、tnm分期及血清afp水平正相關(guān),與患者預后顯著負相關(guān)性。結(jié)論:1.營養(yǎng)缺乏環(huán)境促進肝癌細胞線粒體融合。2.pka介導的drp1磷酸化(s637位點)促進營養(yǎng)缺乏時的線粒體融合。3.線粒體融合通過使線粒體嵴形態(tài)重塑調(diào)控肝癌細胞能量代謝并促進肝癌細胞在營養(yǎng)缺乏環(huán)境中存活與腫瘤生長。4.肝癌組織中p-DRP1S637水平是潛在的腫瘤預后標志物。
[Abstract]:Background: mitochondria are the most important organelles regulating cell metabolism. Through continuous division / fusion to regulate their own functions and respond to external stimuli, a large number of studies have confirmed that mitochondrial mitotic / fusion abnormalities are closely related to the development of a variety of neurodegenerative diseases, cardiovascular diseases and metabolic diseases. It is confirmed that mitochondrial mitotic / fusion in various types of tumors is abnormal and participates in malignant progression of tumors. Solid tumors often cause chronic nutritional deficiency due to rapid growth, and tumor cells need to be perceived and adapt through reprogramming to survive, but the mechanism of cancer adaptation to nutritional deficiency is still not ten. It is clear that the mechanism is of great significance to the prevention and control of tumor. Our previous study found that the line particle fusion of hepatoma cells became longer in the absence of nutritional environment. As the core of the cell energy metabolism, it is not clear whether the changes of the morphology and structure of the hepatoma cells are involved in the metabolic adaptation of the liver cancer cells in the nutritional deficiency. Objective: 1. Regulation effect of nutrient deficiency environment on mitochondrial division / fusion of hepatoma cell and mechanism.2. study the role of mitochondrial fusion in regulating the energy metabolism of hepatoma cells.3. analysis of the tumor biological significance of mitochondrial fusion regulating energy metabolism. Methods: 1. using mito-tracker fluorescence staining and transmission electron microscopy (TEM) technology, in hepatoma cell lines and liver Analysis of the regulatory role of nutritional deficiency environment on mitochondrial mitosis and fusion in cancerous tissue specimens.2. uses qRT-PCR and Westernblot experiments to detect the expression and activity of key molecules and upstream signal molecules involved in mitochondrial division / fusion regulation under nutrient deficiency environment, and analyze the diversity of nutrient deficiency environment to promote mitochondrial fusion. Submechanism.3. uses qRT-PCR, Westernblot and GC-MS (gas chromatography-mass spectrometry) technology to analyze the regulation of mitochondrial fusion to glycolytic and oxidative phosphoric acid in liver cancer cells using TEM (transmission electron microscopy), bluenative-page, co-Ip, qRT-PCR and Westernblot techniques, and the regulation of oxidative phosphorylation of mitochondria with mitochondrial fusion when nutrient deficiency is deficient. Study on the mechanism of glycolysis.5. using FCM (flow cytometry) and clone formation experiments to analyze the effect of mitochondrial fusion on the survival of hepatoma cells during nutritional deficiency.6. use nude mouse subcutaneous tumor formation experiments and the analysis of the effects of mitochondrial fusion on tumor growth in vivo.7. use IHC experiment to promote mitochondrial fusion under the nutritional deficiency environment. The expression of key molecules was detected, and its correlation with the clinical parameters and prognosis of liver cancer patients was analyzed. Results: 1.mito-tracker fluorescence staining proved that nutritional deficiency promoted the mitochondrial fusion of hepatoma cells to grow, and the transmission electron microscopy (TEM) observed the mitochondrial morphology of liver cancer tissue, and the tumor Central region (nutritional deficiency) was more than the marginal region (cAMP). The mitochondrial fission / fusion regulation key molecules drp1, FIS1, mfn1, Mfn2 and OPA1 have not changed significantly when mitochondrial.2. nutrition deficiency is long, while mitochondrial localization drp1 is reduced by PKA phosphorylation (s637), resulting in mitochondrial long.3. nutrient deficiency, mitochondrial fusion promotes the oxidation phosphoric acid of hepatoma cells and inhibits sugar. When glycolysis.4. nutrition is deficient, mitochondrial fusion can activate oxidative phosphorylation and inhibit glycolysis by activating mitochondrial crista and promoting oxidative phosphorylation and inhibiting glycolysis. Mitochondrial fusion inhibits glycolytic.5. mitochondrial fusion through oxidative phosphorylation mediated nad+/sirt1/hif-1 alpha signal and inhibits hepatoma cells in the nutritional deficiency environment. Apoptosis, promoting the clonogenic ability of hepatoma cells,.6. nutritional deficiency induced mitochondrial fusion, promoting tumor growth.7. mediated mitochondrial fusion, p-drp1s937 expression level is positively correlated with tumor classification, TNM staging and serum AFP levels, and significantly negatively correlated with the prognosis of patients. Conclusion: 1. nutritional deficiency Mitochondrial fusion.2.pka mediated drp1 phosphorylation (s637 site) promotes mitochondrial fusion of mitochondrial fusion in nutritional deficiency by promoting mitochondrial fusion through mitochondrial crista remodeling to regulate the energy metabolism of hepatoma cells and promote the survival of hepatoma cells in the nutritional deficiency environment and p-DRP1S637 water in.4. liver cancer tissues in the nutritional deficiency environment. Leveling is a potential prognostic marker of tumor.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R735.7

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