自噬在酒精致PC12细胞损伤中的保护作用

发布时间：2018-02-14 01:09

本文关键词： 自噬酒精 PC12 细胞损伤凋亡　出处：《河南大学》2012年硕士论文　论文类型：学位论文

【摘要】：自噬是广泛存在于真核细胞中的生命现象，是真核细胞内降解胞质细胞器和长寿命蛋白质的一种溶酶体途径。它是一个贯穿大分子、核糖体和细胞器被降解过程中高度保守的机制。自噬通常被认为是在饥饿情况下为细胞提供了暂时生存的机会，这些情况包括氨基酸和营养物的缺失、缺氧和代谢应激。程序性细胞死亡是区别去细胞坏死的一种细胞死亡方式，，分为Ⅰ型即凋亡性细胞死亡和Ⅱ型即自噬性细胞死亡。有研究表明，自噬作为一种程序性细胞死亡的方式，在某些情况下，如凋亡阻断，可能将细胞带至最终的死亡。酒精是一种亲神经物质，对人体许多系统、器官均有损伤作用，其中神经系统是其损伤的主要靶器官之一。酒精的神经毒性作用主要表现在氧化损伤、改变细胞质膜的状态、引起细胞凋亡等几个方面。目前国内外对酒精致神经细胞损伤在自噬性死亡方面的研究尚为鲜见。目的：本文以大鼠嗜铬神经瘤细胞PC12为靶细胞，并将PC12细胞分为四组：正常对照组、酒精处理组、酒精+自噬抑制剂处理组、酒精+凋亡抑制剂处理组。以探讨自噬在酒精致PC12细胞损伤过程中的保护作用，为揭示酒精的神经毒性作用的进一步研究提供实验依据，也为酒精致神经细胞损伤的修复提供理论依据。方法：构建重组pEGFP-LC3表达载体，并瞬时转染PC12细胞，以鉴定酒精致PC12细胞损伤过程中自噬的发生；MTT法检测自噬在酒精抑制PC12细胞增殖过程中所起的作用；通过高内涵活细胞成像系统测定活性氧水平来检测自噬在酒精致PC12细胞氧化损伤作用中所起的作用；通过免疫荧光、高内涵活细胞成像系统、Western blot检测酒精致PC12细胞损伤过程中，自噬与凋亡的相互关系。结果：（1）在酒精致PC12细胞损伤过程中有自噬的发生；（2）在酒精致PC12细胞损伤过程中，抑制自噬会导致细胞生存率明显下降，同时细胞的氧化损伤水平显著升高；（3）在酒精致PC12细胞损伤过程中，抑制自噬，细胞Caspase介导的凋亡水平升高，抑制Caspase介导的凋亡，细胞的自噬水平升高。结论：（1）酒精致PC12细胞损伤过程中有自噬发生并起保护作用，维持细胞的稳态平衡；（2）在酒精致PC12细胞损伤中，自噬和凋亡有相关性，抑制自噬可以细胞Caspase介导的凋亡水平升高，抑制Caspase介导的凋亡则细胞自噬水平升高。
[Abstract]:Autophagy is a widespread living phenomenon in eukaryotic cells. It is a lysosomal pathway for degradation of cytoplasmic organelles and long-lived proteins in eukaryotic cells. Highly conservative mechanisms in the degradation of ribosomes and organelles. Autophagy is often thought to provide a temporary survival opportunity for cells under starvation, including the loss of amino acids and nutrients. Anoxia and metabolic stress. Programmed cell death is a cell death pattern that distinguishes acellular necrosis. It is divided into two types: apoptotic cell death and autophagic cell death. Autophagy, as a programmed form of cell death, in some cases, such as blocking apoptosis, may bring cells to eventual death. Alcohol is a neurophilic substance that damages many systems and organs of the human body. Among them, the nervous system is one of the main target organs of its injury. The neurotoxicity of alcohol is mainly manifested in oxidative damage and changes in the state of the cytoplasmic membrane. At present, there are few studies on the effects of alcohol refined nerve cell injury on autophagic death. Objective: to divide rat pheochromocytoma cells (PC12) into four groups: normal control group, alcohol treatment group, alcohol autophagy inhibitor group. In order to explore the protective effect of autophagy on the injury of wine refined PC12 cells, the protective effect of alcohol apoptosis inhibitor was studied in order to provide experimental evidence for further study on the neurotoxic effect of alcohol. It also provides a theoretical basis for the repair of wine refined nerve cell injury. Methods: recombinant pEGFP-LC3 expression vector was constructed and transient transfection of PC12 cells was carried out to identify the role of autophagy in alcohol inhibition of PC12 cell proliferation. The role of autophagy in the oxidative damage of wine refined PC12 cells was detected by high intension living cell imaging system, and the process of wine refined PC12 cell injury was detected by high intension living cell imaging system and immunofluorescence, high intension living cell imaging system and Western blot. The relationship between autophagy and apoptosis. Results (1) autophagy occurred during the injury of wine refined PC12 cells. (2) inhibition of autophagy resulted in a significant decrease in cell survival rate during the injury of wine refined PC12 cells. At the same time, the oxidative damage level of the cells was significantly increased. (3) during the injury of wine refined PC12 cells, the levels of autophagy, Caspase mediated apoptosis, Caspase mediated apoptosis and autophagy levels were inhibited. Conclusion: 1) autophagy may play a protective role in the injury of wine refined PC12 cells, and maintain the homeostasis of the cells. There is a correlation between autophagy and apoptosis in the injury of wine refined PC12 cells, and inhibit the increase of apoptosis induced by Caspase. Inhibition of Caspase-mediated apoptosis increased autophagy levels.
【学位授予单位】：河南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R346

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