本文選題：睡眠剝奪 + 氧化應(yīng)激�。� 參考：《河北醫(yī)科大學(xué)》2015年碩士論文

【摘要】：目的:睡眠在人類生活中占有很重要的位置。睡眠剝奪(sleep deprivation,SD)是指由于現(xiàn)代生活及工作上的需要、周圍環(huán)境或其他無法避免的原因而不能保證正常的睡眠。睡眠剝奪對人類認(rèn)知功能的影響是不容忽視的,包括清醒度、警覺性和注意力下降;學(xué)習(xí)記憶能力降低;執(zhí)行功能下降;負(fù)面情緒增加等。睡眠缺失引起的這些一系列認(rèn)知功能的障礙,與睡眠缺失導(dǎo)致腦部的某些重要的功能區(qū)域及有關(guān)其功能的蛋白質(zhì)的改變密切相關(guān)。睡眠缺失后可以在海馬區(qū)觀察到很多變化如神經(jīng)元的更換、凋亡,樹突和突觸的重構(gòu),在嚙齒類動物的前額葉皮層及杏仁核也可以觀察到其他一些適應(yīng)性改變。在睡眠剝奪后海馬神經(jīng)元有明顯損傷,神經(jīng)元形態(tài)的改變直接影響其正常生理功能的發(fā)揮,睡眠剝奪可引起大鼠海馬神經(jīng)元凋亡發(fā)生。海馬是位于腦顳葉內(nèi)的一個(gè)部位,它是組成大腦邊緣系統(tǒng)的一部分,擔(dān)當(dāng)著關(guān)于記憶以及空間定位的作用。海馬亦是老年期癡呆患者較早發(fā)生病理改變的部位。有研究認(rèn)為睡眠剝奪與很多神經(jīng)退行性疾病密切相關(guān),而多種神經(jīng)退行性疾病均發(fā)現(xiàn)有某些致病蛋白質(zhì)的聚集,由此可知,直接對睡眠剝奪后腦組織的蛋白質(zhì)組變化的研究可更加直觀的揭示其引起的機(jī)體各系統(tǒng)變化尤其是神經(jīng)系統(tǒng)改變的病理生理機(jī)制。蛋白質(zhì)是生物體基因功能活動的最終執(zhí)行者,并直接體現(xiàn)生命現(xiàn)象的復(fù)雜性以及多樣性。蛋白質(zhì)組學(xué)技術(shù)的興起是進(jìn)入后基因組時(shí)代的標(biāo)志,它是從整體的角度來研究蛋白質(zhì)的特征,包括蛋白質(zhì)的表達(dá)水平、翻譯后修飾、蛋白與蛋白之間的作用等,并揭示蛋白質(zhì)功能與細(xì)胞生命現(xiàn)象之間的規(guī)律。差異蛋白質(zhì)組學(xué)則是分析不同生物體在不同狀態(tài)下蛋白質(zhì)表達(dá)的差異,為探索細(xì)胞的分子機(jī)制、疾病的病理病因及尋找治療藥物靶點(diǎn)提供了可能性的依據(jù)。本研究通過對大鼠睡眠剝奪模型的制作,對正常對照組海馬組織和睡眠剝奪72小時(shí)后海馬組織進(jìn)行差異蛋白質(zhì)組學(xué)研究,找出表達(dá)不同的蛋白質(zhì),從而探尋睡眠剝奪后引起一系列的蛋白質(zhì)改變,從而幫助我們進(jìn)一步深入理解這些蛋白質(zhì)改變引起一系列疾病的本質(zhì)。方法:1實(shí)驗(yàn)分組:成年雄性Wistar大鼠分為實(shí)驗(yàn)組和對照組,實(shí)驗(yàn)組大鼠進(jìn)行72小時(shí)睡眠剝奪,對照組大鼠維持正常睡眠-覺醒周期。2制作大鼠睡眠剝奪模型,實(shí)驗(yàn)組大鼠于睡眠剝奪72小時(shí)后處死,斷頭取腦,分離大腦海馬組織;對照組同樣取相應(yīng)部位腦組織,將組織放EP管中后置于-80℃冰箱中備用。3進(jìn)行大鼠海馬組織蛋白質(zhì)提取并使用胰蛋白酶酶解脫鹽后待用。4將制備好的樣品肽段用TMT標(biāo)記,上樣buffer溶解,膠條水化,并經(jīng)TFA酸化后進(jìn)行C18 Zip-Tip脫鹽。5抽干樣品后溶解、離心,并用LC-LTQ-MS/MS分析以及質(zhì)譜數(shù)據(jù)檢索。結(jié)果:1成功制作大鼠睡眠剝奪模型,運(yùn)用蛋白質(zhì)組學(xué)及TMT技術(shù),比較睡眠剝奪72小時(shí)組與正常對照組海馬蛋白質(zhì)的變化,得出差異蛋白99個(gè),其中表達(dá)上調(diào)的蛋白有65個(gè),表達(dá)下調(diào)的蛋白有34個(gè)。2運(yùn)用Gene Codis軟件分析發(fā)現(xiàn)有意義的蛋白60多個(gè),這些蛋白主要涉及到蛋白質(zhì)轉(zhuǎn)運(yùn)及降解、突觸再生、氧化應(yīng)激、細(xì)胞周期、能量代謝與細(xì)胞凋亡等生物學(xué)過程。結(jié)論:1運(yùn)用蛋白質(zhì)組學(xué)及TMT標(biāo)記技術(shù)鑒定出大鼠睡眠剝奪后與對照組海馬組織相比差異表達(dá)蛋白涉及蛋白質(zhì)加工與降解、突觸再生及其功能、氧化應(yīng)激反應(yīng)、細(xì)胞凋亡過程等等,而這些過程多數(shù)與神經(jīng)系統(tǒng)退行性疾病尤其是AD發(fā)生的病理機(jī)制密切相關(guān),提示睡眠缺失與腦老化及神經(jīng)系統(tǒng)退行性疾病可能存在潛在的聯(lián)系。從而幫助我們進(jìn)一步深入理解這些疾病的本質(zhì)。2通過本實(shí)驗(yàn)表明睡眠剝奪導(dǎo)致大腦海馬組織相關(guān)功能蛋白發(fā)生改變,這些蛋白參與神經(jīng)系統(tǒng)退行性疾病的發(fā)生與發(fā)展,進(jìn)一步表明睡眠剝奪與神經(jīng)系統(tǒng)退行性疾病相關(guān)。而退行性疾病是一個(gè)慢性過程,本實(shí)驗(yàn)睡眠剝奪作為一個(gè)急性應(yīng)激因素,去除這一應(yīng)激后即恢復(fù)睡眠后蛋白質(zhì)是否會發(fā)生改變?nèi)圆皇智宄?因此睡眠剝奪與神經(jīng)系統(tǒng)退行性疾病之間的關(guān)系仍須進(jìn)一步研究證實(shí)。
[Abstract]:Objective: sleep occupies an important position in human life. Sleep deprivation (SD) refers to the lack of normal sleep due to the needs of modern life and work, the surrounding environment or other unavoidable reasons. The effect of sleep deprivation on human cognitive function is not to be ignored, including sober, vigilance, and the effect of sleep deprivation. Decline in attention, reduced learning and memory ability, decline in executive function, negative emotion, and a series of cognitive impairment caused by lack of sleep, which are closely related to some important functional areas of the brain and the changes in the proteins associated with their functions. Changes such as replacement of neurons, apoptosis, dendrites and synapses can also be observed in other adaptive changes in the prefrontal cortex and amygdala of rodents. After sleep deprivation, hippocampal neurons have obvious damage. Changes in neuron morphology directly affect their normal physiological functions. Sleep deprivation can cause rats. Hippocampus is a part of the hippocampus, a part of the brain's temporal lobe, a part of the cerebral marginal system that plays a role in memory and spatial localization. Hippocampus is also an early pathological change in Alzheimer's disease. A variety of neurodegenerative diseases have found the aggregation of some pathogenic proteins. Therefore, the study of the changes in the protein group directly after sleep deprivation can more directly reveal the pathophysiological mechanism of the changes in the system, especially the changes in the nervous system. Protein is the functional activity of the organism. The ultimate executor embodies the complexity and diversity of the life phenomenon. The rise of proteomics technology is the symbol of the post genome era. It is a whole to study the characteristics of protein, including the expression level of protein, the post translation modification, the role of protein and protein, and reveal the function of protein and the function of protein. Differential proteomics is the analysis of the difference in protein expression between different organisms in different states, which provides the basis for exploring the molecular mechanism of the cells, the pathogeny of the disease and finding the therapeutic target for the treatment of the drug. After 72 hours of hippocampal and sleep deprivation, the differential proteomic study of hippocampal tissue was conducted to find different proteins and to explore a series of protein changes after sleep deprivation, which helped us further understand the nature of the protein changes that caused a series of diseases. Method: 1 experimental groups: adult: Adult The male Wistar rats were divided into the experimental group and the control group. The rats in the experimental group were deprived of sleep for 72 hours. The control group maintained normal sleep awakening cycle.2 to make the rat sleep deprivation model. The rats in the experimental group died after 72 hours of sleep deprivation, took the head to take the brain, and separated the brain tissue. The control group also took the corresponding brain tissue in the group. After being placed in the EP tube in the -80 centigrade, the protein extracted from the hippocampus of the rat was extracted from the refrigerator in the refrigerator of -80 C and then used by the trypsinase to remove the salt. The prepared sample peptide was marked with TMT, the sample was dissolved by buffer, the glue strip was hydrated, and after TFA acidification, the C18 Zip-Tip desalination.5 was dissolved, centrifuged, and used LC-LTQ-MS/MS analysis. Results: 1 the result: 1 successfully made the rat sleep deprivation model, using proteomics and TMT technology, compared the changes in the protein of the hippocampus in the 72 hour sleep deprivation group and the normal control group, and obtained the difference protein 99, of which there were 65 up-regulated proteins and 34.2 expressed by Gene Codis software to analyze hair. The existing more than 60 proteins are mainly involved in biological processes such as protein transport and degradation, synapse regeneration, oxidative stress, cell cycle, energy metabolism and cell apoptosis. Conclusion: 1 using proteomics and TMT labeling technique, the differential expression protein of rat after sleep deprivation was identified as compared with the control group. Protein processing and degradation, synapse regeneration and its function, oxidative stress response, apoptosis process, and so on, most of these processes are closely related to the pathological mechanism of the neurodegenerative disease, especially the AD, suggesting that there is a potential link between sleep loss and brain aging and neurodegenerative diseases. An in-depth understanding of the nature of these diseases.2 through this experiment shows that sleep deprivation leads to changes in functional proteins associated with the brain's hippocampus, which are involved in the occurrence and development of neurodegenerative diseases, and further indicate that sleep deprivation is associated with neurodegenerative diseases. Degenerative disease is a chronic process, In this experiment, sleep deprivation as an acute stress factor is not very clear after the removal of this stress, that is, whether the protein will change after sleep. Therefore, the relationship between sleep deprivation and neurodegenerative diseases must be further studied.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R740

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