脂代謝重編程在宮頸癌放療抵抗中的作用及分子機(jī)制研究
發(fā)布時(shí)間：2018-10-14 11:33
【摘要】：背景宮頸癌(cervical cancer)是嚴(yán)重威脅婦女健康的常見惡性腫瘤之一,占全球女性的第三位。近年來發(fā)病率不斷上升,大約每年有460,000新發(fā)病例,其中約274,000萬患者死亡,且發(fā)病逐漸年輕化。放射治療在宮頸癌的治療中具有極其重要作用和地位,尤其對(duì)于已失去手術(shù)機(jī)會(huì)的中晚期及復(fù)發(fā)宮頸癌,放射治療是其主要的治療手段。近年來,雖然隨著放療技術(shù)的發(fā)展及放療設(shè)備的優(yōu)化,其臨床治療效果有了一定程度的提高,但總體療效仍不樂觀,放療抵抗是治療失敗的主要原因。因此,探討宮頸癌放療抵抗的機(jī)制,提高其放療敏感性對(duì)于其治療具有十分重要臨床意義。代謝重編程在腫瘤的發(fā)生發(fā)展中發(fā)揮重要作用。既往對(duì)于代謝重編程在腫瘤生物學(xué)特性中的研究表明其可誘導(dǎo)腫瘤細(xì)胞凋亡耐受、促進(jìn)腫瘤侵襲轉(zhuǎn)移以及化療耐受.但代謝重編程是否參與宮頸癌放療抵抗尚不明確。線粒體是細(xì)胞的能量供應(yīng)的主要場(chǎng)所,維持線粒體功能正常對(duì)確保細(xì)胞在應(yīng)激狀態(tài)下能量供應(yīng)充足而對(duì)抗凋亡具有重要意義。線粒體氧化磷酸化過程中釋放ROS導(dǎo)致線粒體損傷,受損線粒體釋放細(xì)胞色素C及Caspase等誘導(dǎo)細(xì)胞凋亡。因此有效清除受損線粒體是維持線粒體功能正常的重要保障。既往研究已經(jīng)發(fā)現(xiàn),放療可誘導(dǎo)腦膠質(zhì)瘤細(xì)胞自噬發(fā)生,自噬抑制劑(3-MA)能顯著增強(qiáng)乳腺癌MCF-7細(xì)胞對(duì)放射線的敏感性,表明自噬在細(xì)胞對(duì)放射線的反應(yīng)中起保護(hù)作用。放射線誘導(dǎo)細(xì)胞自噬,增加He La及H1299細(xì)胞放療敏感性。但線粒體自噬在宮頸癌的放療抵抗中的作用及其機(jī)制尚不明確。因此本課題擬在前期宮頸癌放療抵抗細(xì)胞株的建立基礎(chǔ)上,探究宮頸癌放療抵抗的能量代謝基礎(chǔ)及線粒體自噬在在宮頸癌的放療抵抗中的作用及機(jī)制。目的1.明確宮頸癌放療抵抗細(xì)胞株的能量代謝特點(diǎn);2.證實(shí)線粒體自噬在宮頸癌放療抵抗中的作用;3.探討代謝重編程調(diào)控線粒體自噬介導(dǎo)宮頸癌放療抵抗的分子機(jī)制。材料與方法1.采用基因芯片分析放宮頸癌Hela及Hela-R細(xì)胞的基因表達(dá)譜;采用Western-blot等方法,檢測(cè)Hela及Hela-R細(xì)胞中代謝酶的表達(dá)特點(diǎn)及其相關(guān)性。2.采用代謝組學(xué)及脂質(zhì)組學(xué)分析Hela及Hela-R細(xì)胞的代謝特點(diǎn),seahorse檢測(cè)兩組細(xì)胞線粒體功能(OCAR)及糖酵解能力(ECAR)。采用激光共聚焦和透射電鏡,觀察射治療條件下宮頸癌細(xì)胞的線粒體膜電位(Δψ)及線粒體自噬情況;4.慢病毒載體建立CRLS1敲低及過表達(dá)的細(xì)胞模型用于研究CRLS1對(duì)宮頸癌放療抵抗的作用及機(jī)制。實(shí)驗(yàn)結(jié)果1.基因芯片、Western-blot及代謝組學(xué)結(jié)果表明,Hela與Hela-R細(xì)胞相比,糖酵解無明顯差異,而線粒體內(nèi)脂質(zhì)氧化明顯增強(qiáng)2.Mitotracker及seahorse結(jié)果表明,Hela-R細(xì)胞的線粒體功能明顯高于其親本Hela細(xì)胞。3.電鏡及激光共聚焦結(jié)果表明,放療抵抗細(xì)胞株在接受放射線照射后線粒體自噬明顯高于親本4.CRLS1表達(dá)上調(diào)增加線粒體心磷脂從而增強(qiáng)線粒體自噬。主要結(jié)論1.宮頸癌放療抵抗過程中發(fā)生了脂代謝重編程。2.線粒體自噬增強(qiáng)是宮頸癌放療抵抗細(xì)胞線粒體功能增加的主要原因。3.CRLS1介導(dǎo)心磷脂合成誘導(dǎo)線粒體自噬促進(jìn)宮頸癌放療抵抗。
[Abstract]:Background cervical cancer is one of the most common malignant tumors that seriously threaten the health of women, accounting for the third place in global women. In recent years, there has been a rising incidence of about 460,000 new cases per year, of which some 274,000,000 patients die and the onset is younger. Radiotherapy plays an important role in the treatment of cervical cancer, especially for the advanced and recurrent cervical cancer that have lost the chance of surgery. Radiotherapy is the main treatment method. In recent years, although with the development of radiotherapy technology and the optimization of radiotherapy equipment, its clinical therapeutic effect has been improved to a certain extent, but overall curative effect is still not optimistic, radiation therapy resistance is the main cause of treatment failure. Therefore, it is very important to study the mechanism of radiotherapy resistance of cervical cancer and improve the radiosensitivity of cervical cancer. Metabolic reprogramming plays an important role in the development of tumor. Previous studies on metabolic reprogramming in tumor biology have shown that it can induce tumor cell apoptosis tolerance, promote tumor invasion and metastasis, and chemotherapy tolerance. However, it is not clear whether metabolic reprogramming is involved in the treatment of cervical cancer radiotherapy. mitochondria are the main sites for the energy supply of cells, and it is of great significance to maintain the normal mitochondrial function and to ensure that the cells are adequately supplied with sufficient energy in a nuclear state. The release of ROS during mitochondrial oxidative phosphorylation led to mitochondrial damage, damaged mitochondria release cytochrome C and Caspase-induced apoptosis. Therefore, the effective clearance of damaged mitochondria is an important guarantee to maintain the function of mitochondria. Previous studies have found that radiotherapy can induce autophagy of glioma cells, and autophagy inhibitors (3-MA) can significantly enhance the sensitivity of breast cancer MCF-7 cells to radiation, suggesting that autophagy plays a protective role in response to radiation. Radiation induced cell autophagy increased the radiosensitivity of He La and H1299 cells. But the role and mechanism of mitochondrial autophagy in the treatment of cervical cancer is unclear. Therefore, it is proposed to explore the role and mechanism of energy metabolism and mitochondrial autophagy in the treatment of cervical cancer. Purpose 1. Clarify the energy metabolism characteristics of cervical cancer radiotherapy resistant cell lines; 2. To confirm the role of mitochondrial autophagy in the treatment of cervical cancer radiotherapy; 3. To explore the molecular mechanism of metabolic reprogramming regulating mitochondrial autophagy-mediated radiotherapy resistance of cervical cancer. Materials and Methods 1. Gene expression profiles were analyzed by gene chip analysis and Western-blot. The expression of metabolic enzymes and their correlation were detected by Western-blot. The mitochondrial function (OCAR) and glycolytic ability (ECAR) of two groups of cell mitochondria were detected by using metabolic group and lipid group. The autophagy and autophagy of cervical carcinoma cells were observed by laser confocal microscope and transmission electron microscope (TEM). The slow virus vector established CRLS1 knockdown and over-expressed cell model to study the effect and mechanism of CRLS1 on radiotherapy resistance of cervical cancer. Experimental results 1. The results of gene chip, Western-blot and metabolic group showed that the mitochondrial function of CDR3-R cells was significantly higher than that of its parents. The results showed that there was no significant difference in the sugar content, but the lipid oxidation in mitochondria was enhanced by 2. Mitoracker and seaorse showed that the mitochondrial function of CDR3-R cells was significantly higher than that of its parents. The results of electron microscopy and confocal laser confocal showed that the autophagy of mitochondria was significantly higher than that of parent 4. CRLS1 expression increased mitochondrial core phospholipid to enhance mitochondrial autophagy. Main conclusions 1. Lipid metabolism reprogramming occurred during the treatment of cervical cancer radiotherapy. The autophagy enhancement of mitochondria is the main reason for the increase of mitochondrial function in the radiotherapy of cervical cancer.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R737.33
[Abstract]:Background cervical cancer is one of the most common malignant tumors that seriously threaten the health of women, accounting for the third place in global women. In recent years, there has been a rising incidence of about 460,000 new cases per year, of which some 274,000,000 patients die and the onset is younger. Radiotherapy plays an important role in the treatment of cervical cancer, especially for the advanced and recurrent cervical cancer that have lost the chance of surgery. Radiotherapy is the main treatment method. In recent years, although with the development of radiotherapy technology and the optimization of radiotherapy equipment, its clinical therapeutic effect has been improved to a certain extent, but overall curative effect is still not optimistic, radiation therapy resistance is the main cause of treatment failure. Therefore, it is very important to study the mechanism of radiotherapy resistance of cervical cancer and improve the radiosensitivity of cervical cancer. Metabolic reprogramming plays an important role in the development of tumor. Previous studies on metabolic reprogramming in tumor biology have shown that it can induce tumor cell apoptosis tolerance, promote tumor invasion and metastasis, and chemotherapy tolerance. However, it is not clear whether metabolic reprogramming is involved in the treatment of cervical cancer radiotherapy. mitochondria are the main sites for the energy supply of cells, and it is of great significance to maintain the normal mitochondrial function and to ensure that the cells are adequately supplied with sufficient energy in a nuclear state. The release of ROS during mitochondrial oxidative phosphorylation led to mitochondrial damage, damaged mitochondria release cytochrome C and Caspase-induced apoptosis. Therefore, the effective clearance of damaged mitochondria is an important guarantee to maintain the function of mitochondria. Previous studies have found that radiotherapy can induce autophagy of glioma cells, and autophagy inhibitors (3-MA) can significantly enhance the sensitivity of breast cancer MCF-7 cells to radiation, suggesting that autophagy plays a protective role in response to radiation. Radiation induced cell autophagy increased the radiosensitivity of He La and H1299 cells. But the role and mechanism of mitochondrial autophagy in the treatment of cervical cancer is unclear. Therefore, it is proposed to explore the role and mechanism of energy metabolism and mitochondrial autophagy in the treatment of cervical cancer. Purpose 1. Clarify the energy metabolism characteristics of cervical cancer radiotherapy resistant cell lines; 2. To confirm the role of mitochondrial autophagy in the treatment of cervical cancer radiotherapy; 3. To explore the molecular mechanism of metabolic reprogramming regulating mitochondrial autophagy-mediated radiotherapy resistance of cervical cancer. Materials and Methods 1. Gene expression profiles were analyzed by gene chip analysis and Western-blot. The expression of metabolic enzymes and their correlation were detected by Western-blot. The mitochondrial function (OCAR) and glycolytic ability (ECAR) of two groups of cell mitochondria were detected by using metabolic group and lipid group. The autophagy and autophagy of cervical carcinoma cells were observed by laser confocal microscope and transmission electron microscope (TEM). The slow virus vector established CRLS1 knockdown and over-expressed cell model to study the effect and mechanism of CRLS1 on radiotherapy resistance of cervical cancer. Experimental results 1. The results of gene chip, Western-blot and metabolic group showed that the mitochondrial function of CDR3-R cells was significantly higher than that of its parents. The results showed that there was no significant difference in the sugar content, but the lipid oxidation in mitochondria was enhanced by 2. Mitoracker and seaorse showed that the mitochondrial function of CDR3-R cells was significantly higher than that of its parents. The results of electron microscopy and confocal laser confocal showed that the autophagy of mitochondria was significantly higher than that of parent 4. CRLS1 expression increased mitochondrial core phospholipid to enhance mitochondrial autophagy. Main conclusions 1. Lipid metabolism reprogramming occurred during the treatment of cervical cancer radiotherapy. The autophagy enhancement of mitochondria is the main reason for the increase of mitochondrial function in the radiotherapy of cervical cancer.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R737.33
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