脂代谢重编程在宫颈癌放疗抵抗中的作用及分子机制研究

发布时间：2018-10-14 11:33

【摘要】：背景宫颈癌(cervical cancer)是严重威胁妇女健康的常见恶性肿瘤之一,占全球女性的第三位。近年来发病率不断上升,大约每年有460,000新发病例,其中约274,000万患者死亡,且发病逐渐年轻化。放射治疗在宫颈癌的治疗中具有极其重要作用和地位,尤其对于已失去手术机会的中晚期及复发宫颈癌,放射治疗是其主要的治疗手段。近年来,虽然随着放疗技术的发展及放疗设备的优化,其临床治疗效果有了一定程度的提高,但总体疗效仍不乐观,放疗抵抗是治疗失败的主要原因。因此,探讨宫颈癌放疗抵抗的机制,提高其放疗敏感性对于其治疗具有十分重要临床意义。代谢重编程在肿瘤的发生发展中发挥重要作用。既往对于代谢重编程在肿瘤生物学特性中的研究表明其可诱导肿瘤细胞凋亡耐受、促进肿瘤侵袭转移以及化疗耐受.但代谢重编程是否参与宫颈癌放疗抵抗尚不明确。线粒体是细胞的能量供应的主要场所,维持线粒体功能正常对确保细胞在应激状态下能量供应充足而对抗凋亡具有重要意义。线粒体氧化磷酸化过程中释放ROS导致线粒体损伤,受损线粒体释放细胞色素C及Caspase等诱导细胞凋亡。因此有效清除受损线粒体是维持线粒体功能正常的重要保障。既往研究已经发现,放疗可诱导脑胶质瘤细胞自噬发生,自噬抑制剂(3-MA)能显著增强乳腺癌MCF-7细胞对放射线的敏感性,表明自噬在细胞对放射线的反应中起保护作用。放射线诱导细胞自噬,增加He La及H1299细胞放疗敏感性。但线粒体自噬在宫颈癌的放疗抵抗中的作用及其机制尚不明确。因此本课题拟在前期宫颈癌放疗抵抗细胞株的建立基础上,探究宫颈癌放疗抵抗的能量代谢基础及线粒体自噬在在宫颈癌的放疗抵抗中的作用及机制。目的1.明确宫颈癌放疗抵抗细胞株的能量代谢特点;2.证实线粒体自噬在宫颈癌放疗抵抗中的作用;3.探讨代谢重编程调控线粒体自噬介导宫颈癌放疗抵抗的分子机制。材料与方法1.采用基因芯片分析放宫颈癌Hela及Hela-R细胞的基因表达谱;采用Western-blot等方法,检测Hela及Hela-R细胞中代谢酶的表达特点及其相关性。2.采用代谢组学及脂质组学分析Hela及Hela-R细胞的代谢特点,seahorse检测两组细胞线粒体功能(OCAR)及糖酵解能力(ECAR)。采用激光共聚焦和透射电镜,观察射治疗条件下宫颈癌细胞的线粒体膜电位(Δψ)及线粒体自噬情况;4.慢病毒载体建立CRLS1敲低及过表达的细胞模型用于研究CRLS1对宫颈癌放疗抵抗的作用及机制。实验结果1.基因芯片、Western-blot及代谢组学结果表明,Hela与Hela-R细胞相比,糖酵解无明显差异,而线粒体内脂质氧化明显增强2.Mitotracker及seahorse结果表明,Hela-R细胞的线粒体功能明显高于其亲本Hela细胞。3.电镜及激光共聚焦结果表明,放疗抵抗细胞株在接受放射线照射后线粒体自噬明显高于亲本4.CRLS1表达上调增加线粒体心磷脂从而增强线粒体自噬。主要结论1.宫颈癌放疗抵抗过程中发生了脂代谢重编程。2.线粒体自噬增强是宫颈癌放疗抵抗细胞线粒体功能增加的主要原因。3.CRLS1介导心磷脂合成诱导线粒体自噬促进宫颈癌放疗抵抗。
[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.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.33

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