本文關(guān)鍵詞： 自噬 酒精 PC12 細(xì)胞損傷 凋亡　出處：《河南大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：自噬是廣泛存在于真核細(xì)胞中的生命現(xiàn)象，是真核細(xì)胞內(nèi)降解胞質(zhì)細(xì)胞器和長壽命蛋白質(zhì)的一種溶酶體途徑。它是一個(gè)貫穿大分子、核糖體和細(xì)胞器被降解過程中高度保守的機(jī)制。自噬通常被認(rèn)為是在饑餓情況下為細(xì)胞提供了暫時(shí)生存的機(jī)會(huì)，這些情況包括氨基酸和營養(yǎng)物的缺失、缺氧和代謝應(yīng)激。程序性細(xì)胞死亡是區(qū)別去細(xì)胞壞死的一種細(xì)胞死亡方式，，分為Ⅰ型即凋亡性細(xì)胞死亡和Ⅱ型即自噬性細(xì)胞死亡。有研究表明，自噬作為一種程序性細(xì)胞死亡的方式，在某些情況下，如凋亡阻斷，可能將細(xì)胞帶至最終的死亡。酒精是一種親神經(jīng)物質(zhì)，對(duì)人體許多系統(tǒng)、器官均有損傷作用，其中神經(jīng)系統(tǒng)是其損傷的主要靶器官之一。酒精的神經(jīng)毒性作用主要表現(xiàn)在氧化損傷、改變細(xì)胞質(zhì)膜的狀態(tài)、引起細(xì)胞凋亡等幾個(gè)方面。目前國內(nèi)外對(duì)酒精致神經(jīng)細(xì)胞損傷在自噬性死亡方面的研究尚為鮮見。 目的：本文以大鼠嗜鉻神經(jīng)瘤細(xì)胞PC12為靶細(xì)胞，并將PC12細(xì)胞分為四組：正常對(duì)照組、酒精處理組、酒精+自噬抑制劑處理組、酒精+凋亡抑制劑處理組。以探討自噬在酒精致PC12細(xì)胞損傷過程中的保護(hù)作用，為揭示酒精的神經(jīng)毒性作用的進(jìn)一步研究提供實(shí)驗(yàn)依據(jù)，也為酒精致神經(jīng)細(xì)胞損傷的修復(fù)提供理論依據(jù)。 方法：構(gòu)建重組pEGFP-LC3表達(dá)載體，并瞬時(shí)轉(zhuǎn)染PC12細(xì)胞，以鑒定酒精致PC12細(xì)胞損傷過程中自噬的發(fā)生；MTT法檢測自噬在酒精抑制PC12細(xì)胞增殖過程中所起的作用；通過高內(nèi)涵活細(xì)胞成像系統(tǒng)測定活性氧水平來檢測自噬在酒精致PC12細(xì)胞氧化損傷作用中所起的作用；通過免疫熒光、高內(nèi)涵活細(xì)胞成像系統(tǒng)、Western blot檢測酒精致PC12細(xì)胞損傷過程中，自噬與凋亡的相互關(guān)系。 結(jié)果：（1）在酒精致PC12細(xì)胞損傷過程中有自噬的發(fā)生；（2）在酒精致PC12細(xì)胞損傷過程中，抑制自噬會(huì)導(dǎo)致細(xì)胞生存率明顯下降，同時(shí)細(xì)胞的氧化損傷水平顯著升高；（3）在酒精致PC12細(xì)胞損傷過程中，抑制自噬，細(xì)胞Caspase介導(dǎo)的凋亡水平升高，抑制Caspase介導(dǎo)的凋亡，細(xì)胞的自噬水平升高。 結(jié)論：（1）酒精致PC12細(xì)胞損傷過程中有自噬發(fā)生并起保護(hù)作用，維持細(xì)胞的穩(wěn)態(tài)平衡；（2）在酒精致PC12細(xì)胞損傷中，自噬和凋亡有相關(guān)性，抑制自噬可以細(xì)胞Caspase介導(dǎo)的凋亡水平升高，抑制Caspase介導(dǎo)的凋亡則細(xì)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.
【學(xué)位授予單位】：河南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R346

【參考文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 石貞玉;酒精致PC12細(xì)胞自噬的研究[D];河南大學(xué);2010年

本文編號(hào)：1509528