【摘要】：線粒體鈣穩(wěn)態(tài)對線粒體功能及細胞的影響至關重要,線粒體鈣維持胞漿鈣離子濃度動態(tài)平衡,是線粒體呼吸功能的重要調節(jié)因素,鈣離子失衡導致細胞凋亡。線粒體鈣穩(wěn)態(tài)重塑是多種疾病發(fā)生的主要原因,但這一改變是否在腫瘤惡性進展中發(fā)揮關鍵作用尚無明確報道。我們的研究團隊首次發(fā)現:新近報道的線粒體鈣離子攝取關鍵調節(jié)分子MCUR1,促進肝癌細胞粒體鈣穩(wěn)態(tài)攝取,其表達水平在肝癌組織細胞中異常升高,MCUR1高表達能夠通過加速細胞增殖抑制線粒體依賴的內源性細胞凋亡而促進了肝癌細胞的生長能力,并且與肝癌病人的不良預后相關。文獻報道,線粒體的鈣離子環(huán)境是調控細胞活性氧ROS生成的關鍵因素,而活性氧ROS作為胞內重要的第二信使,能夠激活包括與腫瘤細胞增殖,凋亡,轉移與腫瘤細胞的血管生成相關的多種關鍵信號通路,最終導致惡性腫瘤發(fā)生。我們進一步研究發(fā)現MCUR1介導的線粒體鈣穩(wěn)態(tài)重塑導致肝癌細胞活性氧ROS水平上調,這一改變與其下游P53降解,AKT/MDM2信號通路活化密切相關。基于此,我們提出假說:MCUR1上調引發(fā)線粒體鈣穩(wěn)態(tài)重塑,通過調控胞內第二信使ROS生成影響其下游akt/mdm2介導的p53降解信號通路,最終促進肝癌的惡性進展。【目的】本課題以肝癌為研究對象,選擇線粒體鈣離子攝取關鍵調節(jié)分子mcur1,明確mcur1介導的線粒體鈣穩(wěn)態(tài)重塑是否參與對肝癌生長的調控,并探索mcur1介導的線粒體鈣穩(wěn)態(tài)重塑調控肝癌生長的作用機制。【方法】1.利用免疫組化實驗分析mcur1以及p53在原發(fā)性肝癌臨床標本的表達及相關性。2.利用mts法測定細胞生長曲線、克隆形成實驗、annexin-v凋亡檢測、流式細胞周期檢測、edu細胞增殖等實驗方法,分析mcur1對肝癌細胞增殖凋亡及生長的影響。3.利用裸鼠皮下荷瘤實驗,分析mcur1在體內對肝癌生長的影響。4.利用rhod-2,fluo-4活細胞染色,分析mcur1對肝癌細胞線粒體基礎鈣離子水平以及線粒體對胞漿鈣的緩沖作用的影響。5.利用dcf及mitosox染料對細胞ros及線粒體ros進行染色,分析mcur1對肝癌細胞線粒體ros生成的影響。6.利用westernblot檢測凋亡相關分子,以及增殖相關分子的表達改變,分析mcur1介導的線粒體鈣穩(wěn)態(tài)重塑促進肝癌細胞的生長的作用機制�！窘Y果】第一部分:通過對原發(fā)性肝癌臨床標本進行分析,發(fā)現mcur1在肝癌組織中表達上調,與肝癌不良預后相關。通過采用mts法測定細胞生長曲線、克隆形成實驗、annexin-v凋亡檢測、流式細胞周期檢測、edu細胞增殖等實驗方法,發(fā)現肝癌細胞中mcur1通過抑制細胞凋亡并且加速細胞增殖而促進肝癌細胞的生長。通過進行裸鼠皮下荷瘤實驗,證實mcur1通過抑制肝癌凋亡,加速肝癌增殖,而促進肝癌的生長。第二部分:通過rhod-2,fluo-4染色檢測線粒體鈣以及胞漿鈣,發(fā)現:mcur1能夠促進線粒體鈣離子攝入,提升線粒體基礎鈣離子濃度,并且在應急狀態(tài)下線粒體對胞漿鈣的緩沖力。通過對線粒體單向轉運體mcu進行干預處理,發(fā)現mcur1依賴于mcu促進肝癌細胞線粒體鈣的攝取。第三部分:通過對細胞ROS以及線粒體ROS的檢測,發(fā)現MCUR1能夠促進肝癌細胞線粒體ROS生成。通過線粒體的ROS清除劑Mito-TEMPO對細胞中線粒體ROS進行清除,以及線粒體鈣清除載體PV-Mito對線粒體內的鈣離子進行清除,發(fā)現MCUR1介導的線粒體鈣離子穩(wěn)態(tài)重塑通過促進肝癌細胞線粒體ROS生成,而抑制細胞凋亡促進細胞增殖。第四部分:通過檢測凋亡相關分子,增殖相關分子,P53,AKT及MDM2磷酸化的檢測,證實:MCUR1介導的線粒體鈣穩(wěn)態(tài)重塑通過ROS/AKT/MDM2途徑使P53失活促進肝癌細胞生長。【結論】本課題研究發(fā)現:MCUR1介導肝癌線粒體鈣離子攝入增強,重塑的線粒體鈣穩(wěn)態(tài)影響ROS的生成,通過ROS/AKT/MDM2途徑使P53失活,調控肝癌的增殖凋亡,最終促進腫瘤進一步惡化。
[Abstract]:The steady state of mitochondrial calcium is critical to the function of the mitochondria and the effect of the cell, and the dynamic balance of the calcium ion concentration in the mitochondria is an important regulation factor of the mitochondrial respiratory function, and the imbalance of the calcium ions leads to the apoptosis of the cells. The steady-state remodeling of mitochondria is the main cause of various diseases, but it is not clear that this change plays a key role in the development of the malignant tumor. Our team first found that the newly-reported mitochondrial calcium ion uptake key regulatory molecule, MCU1, promotes the steady uptake of calcium and calcium in the liver cancer cell, and the expression level of the mitochondrial calcium ion is abnormally elevated in the liver cell of the liver cancer, The high expression of MCUR1 can promote the growth ability of the liver cancer cells by accelerating the cell proliferation and inhibiting the mitochondria-dependent endogenous cell apoptosis, and is related to the poor prognosis of the liver cancer patients. The literature reports that the calcium ion environment of mitochondria is a key factor to regulate the production of reactive oxygen ROS, and reactive oxygen ROS, as a second messenger in the cell, can activate various key signal pathways involved in the proliferation, apoptosis, metastasis and angiogenesis of tumor cells, Resulting in the occurrence of a malignant tumor. We further study that MCUR1-mediated steady-state remodeling of mitochondrial calcium results in an up-regulation of reactive oxygen ROS level in liver cancer cells, which is closely related to the downstream P53 degradation, AKT/ MDM2 signaling pathway activation. Based on this, we put forward the hypothesis that the MUR1 upregulates the steady-state remodeling of mitochondrial calcium, and by regulating the second messenger ROS in the cell, the downstream akt/ mdm2-mediated p53 degradation signal pathway is generated, and the malignant progression of the liver cancer is finally promoted. [Objective] To study whether the steady-state remodeling of mitochondrial calcium mediated by mcur1 is involved in the regulation of the growth of the liver cancer, and to explore the mechanism of the role of mcur1-mediated steady-state remodeling of the mitochondria in the control of the growth of the liver cancer. [Method] 1. The expression and correlation of mcur1 and p53 in the clinical specimens of primary liver cancer were analyzed by immunohistochemistry. The effects of mcur1 on the proliferation and apoptosis and growth of liver cancer cells were analyzed by using the method of mts to determine cell growth curve, clone formation experiment, annexin-v apoptosis detection, flow cytometry cycle detection and edu cell proliferation. The effect of mcur1 on the growth of liver cancer in vivo was analyzed using the nude mice's subcutaneous tumor-bearing experiment. The effect of mcur1 on the calcium ion level of the mitochondria of the liver cancer cells and the effect of the mitochondria on the calcium in the cytoplasm of the liver cancer cells was analyzed by using the r2005-2 and fluo-4 living cells. The effects of mcur1 on the generation of mitochondria in liver cancer cells were analyzed by using dcf and mittox dyes to dye the cells and the mitochondria. By using the western blot to detect the apoptosis-related molecules and the change of the expression of the related molecules, the mechanism of the role of mcur1-mediated steady-state remodeling of the mitochondrial calcium to promote the growth of the liver cancer cells was analyzed. [Results] The first part: Through the analysis of the clinical specimen of primary liver cancer, it was found that the expression of mcur1 in the liver cancer tissue was up-regulated and related to the bad prognosis of the liver cancer. By using the method of mts to determine the cell growth curve, the clone formation experiment, the annexin-v apoptosis test, the flow cytometry cycle detection, the edu cell proliferation, and the like, the mcur1 in the liver cancer cell is found to promote the growth of the liver cancer cell by inhibiting the cell apoptosis and accelerating the cell proliferation. By carrying out the subcutaneous tumor-bearing experiment of the nude mouse, the mcur1 is confirmed to promote the growth of the liver cancer by inhibiting the apoptosis of the liver cancer and accelerating the proliferation of the liver cancer. In the second part, the mitochondrial calcium and the cytosolic calcium were detected by rF-2 and fluo-4 staining, and it was found that mcur1 can promote the calcium ion uptake of mitochondria, increase the calcium ion concentration of the mitochondria, and reduce the buffering force of the mitochondria to the cytosolic calcium in the emergency state. It was found that mcur1 was dependent on the uptake of mitochondrial calcium in liver cancer cells. The third part: through the detection of ROS and the mitochondrial ROS, it is found that the MCU1 can promote the generation of the mitochondrial ROS in the liver cancer cells. The mitochondrial ROS in the cells was removed by the mitochondrial ROS scavenger, Mito-TEMPO, and the mitochondrial calcium removal vector PV-Mito was removed from the mitochondria, and the MUR1-mediated steady-state remodeling of the mitochondrial calcium ions was found by promoting the generation of mitochondrial ROS in the liver cancer cells, And the cell apoptosis is inhibited and the cell proliferation is promoted. The fourth part: Through the detection of the apoptosis-related molecules, the proliferation-related molecules, the P53, AKT and MDM2 phosphorylation, it was confirmed that the MUR1-mediated steady-state remodeling of the mitochondria promoted the growth of the liver cancer cells through the ROS/ AKT/ MDM2 pathway. [Conclusion] The study found that MCUR1 mediates the increase of mitochondrial calcium ion uptake in liver cancer, and the remodeling of the mitochondrial calcium homeostasis affects the production of ROS, and by means of the ROS/ AKT/ MDM2 pathway, the P53 is inactivated, the proliferation and apoptosis of the liver cancer are regulated, and the further deterioration of the tumor is promoted.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R735.7

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