线粒体融合参与营养缺乏环境肝癌细胞糖代谢重编程调控的作用与机制研究

发布时间：2018-06-11 18:58

本文选题：营养缺乏环境 + 线粒体分裂/融合　；参考：《第四军医大学》2016年博士论文

【摘要】：背景:线粒体是调控细胞代谢最重要的细胞器,通过不断地分裂/融合调控自身功能并对外界刺激做出适应性反应。大量研究证实,线粒体分裂/融合异常与多种神经退行性疾病、心血管疾病及代谢性疾病的发生进展密切相关。近年来研究证实,多种类型肿瘤中线粒体分裂/融合发生异常,并参与肿瘤恶性进展。实体瘤常由于生长过快而导致内部出现慢性营养缺乏微环境,肿瘤细胞需感知并通过代谢重编程做出适应才能继续生存,而目前肿瘤适应营养缺乏的机制仍不十分清楚,阐明其机制对肿瘤防治具有重大意义。我们前期研究发现,营养缺乏环境时肝癌细胞线粒融合变长。作为细胞能量代谢调控核心的线粒体,其形态结构的变化是否参与肝癌细胞在营养缺乏时的代谢适应尚不清楚。目的:1.明确营养缺乏环境对肝癌细胞线粒体分裂/融合的调控作用与机制。2.探讨线粒体融合对肝癌细胞能量代谢的调控作用。3.分析线粒体融合调控能量代谢的肿瘤生物学意义。方法:1.利用mito-tracker荧光染色与透射电镜(tem)技术,在肝癌细胞系与肝癌临床组织标本中分析营养缺乏环境对线粒体分裂与融合的调控作用。2.利用qrt-pcr与westernblot实验,检测营养缺乏环境下参与线粒体分裂/融合调控的关键分子及其上游信号分子的表达与活性,分析营养缺乏环境促进线粒体融合的分子机制。3.利用qrt-pcr、westernblot与gc-ms(气相色谱-质谱联用)技术,分析营养缺乏时线粒体融合对肝癌细胞糖酵解与氧化磷酸的调控。4.利用tem(透射电镜)、bluenative-page、co-ip、qrt-pcr与westernblot技术,对营养缺乏时线粒体融合调控氧化磷酸化与糖酵解的机制进行研究。5.利用fcm(流式细胞术)与克隆形成实验,分析营养缺乏时线粒体融合对肝癌细胞存活的影响。6.利用裸鼠皮下成瘤实验,在体内分析线粒体融合对肿瘤生长的影响。7.利用ihc实验,对促进营养缺乏环境下线粒体融合关键分子的表达进行检测,并对其与肝癌患者临床参数及预后进行相关性分析。结果:1.mito-tracker荧光染色证实营养缺乏促进肝癌细胞线粒体融合变长;透射电镜(tem)对肝癌组织线粒体形态观察发现,肿瘤中心区(营养缺乏)较边缘区(营养充足)线粒体变长。2.营养缺乏时,线粒体分裂/融合调控关键分子drp1、fis1、mfn1、mfn2与opa1表达均未发生显著变化,而线粒体定位的drp1因被pka磷酸化(s637)而减少,导致线粒体变长。3.营养缺乏时,线粒体融合促进肝癌细胞氧化磷酸而抑制糖酵解。4.营养缺乏时,线粒体融合通过使线粒体嵴紧密促进氧化呼吸链复合体组装,从而激活氧化磷酸化而抑制糖酵解;线粒体融合通过氧化磷酸化介导的nad+/sirt1/hif-1α信号抑制糖酵解。5.线粒体融合抑制肝癌细胞在营养缺乏环境下的凋亡,促进肝癌细胞克隆形成能力。6.营养缺乏诱导的线粒体融合促进肿瘤生长。7.介导营养缺乏环境下线粒体融合的关键分子p-drp1s937表达水平与肝癌患者肿瘤分级、tnm分期及血清afp水平正相关,与患者预后显著负相关性。结论:1.营养缺乏环境促进肝癌细胞线粒体融合。2.pka介导的drp1磷酸化(s637位点)促进营养缺乏时的线粒体融合。3.线粒体融合通过使线粒体嵴形态重塑调控肝癌细胞能量代谢并促进肝癌细胞在营养缺乏环境中存活与肿瘤生长。4.肝癌组织中p-DRP1S637水平是潜在的肿瘤预后标志物。
[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.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7

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