本文選題：睡眠剝奪 + 氧化應激�。� 參考：《河北醫(yī)科大學》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;學習記憶能力降低;執(zhí)行功能下降;負面情緒增加等。睡眠缺失引起的這些一系列認知功能的障礙,與睡眠缺失導致腦部的某些重要的功能區(qū)域及有關其功能的蛋白質的改變密切相關。睡眠缺失后可以在海馬區(qū)觀察到很多變化如神經元的更換、凋亡,樹突和突觸的重構,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性改變。在睡眠剝奪后海馬神經元有明顯損傷,神經元形態(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元凋亡發(fā)生。海馬是位于腦顳葉內的一個部位,它是組成大腦邊緣系統(tǒng)的一部分,擔當著關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。有研究認為睡眠剝奪與很多神經退行性疾病密切相關,而多種神經退行性疾病均發(fā)現(xiàn)有某些致病蛋白質的聚集,由此可知,直接對睡眠剝奪后腦組織的蛋白質組變化的研究可更加直觀的揭示其引起的機體各系統(tǒng)變化尤其是神經系統(tǒng)改變的病理生理機制。蛋白質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)象的復雜性以及多樣性。蛋白質組學技術的興起是進入后基因組時代的標志,它是從整體的角度來研究蛋白質的特征,包括蛋白質的表達水平、翻譯后修飾、蛋白與蛋白之間的作用等,并揭示蛋白質功能與細胞生命現(xiàn)象之間的規(guī)律。差異蛋白質組學則是分析不同生物體在不同狀態(tài)下蛋白質表達的差異,為探索細胞的分子機制、疾病的病理病因及尋找治療藥物靶點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小時后海馬組織進行差異蛋白質組學研究,找出表達不同的蛋白質,從而探尋睡眠剝奪后引起一系列的蛋白質改變,從而幫助我們進一步深入理解這些蛋白質改變引起一系列疾病的本質。方法:1實驗分組:成年雄性Wistar大鼠分為實驗組和對照組,實驗組大鼠進行72小時睡眠剝奪,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,實驗組大鼠于睡眠剝奪72小時后處死,斷頭取腦,分離大腦海馬組織;對照組同樣取相應部位腦組織,將組織放EP管中后置于-80℃冰箱中備用。3進行大鼠海馬組織蛋白質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標記,上樣buffer溶解,膠條水化,并經TFA酸化后進行C18 Zip-Tip脫鹽。5抽干樣品后溶解、離心,并用LC-LTQ-MS/MS分析以及質譜數(shù)據(jù)檢索。結果:1成功制作大鼠睡眠剝奪模型,運用蛋白質組學及TMT技術,比較睡眠剝奪72小時組與正常對照組海馬蛋白質的變化,得出差異蛋白99個,其中表達上調的蛋白有65個,表達下調的蛋白有34個。2運用Gene Codis軟件分析發(fā)現(xiàn)有意義的蛋白60多個,這些蛋白主要涉及到蛋白質轉運及降解、突觸再生、氧化應激、細胞周期、能量代謝與細胞凋亡等生物學過程。結論:1運用蛋白質組學及TMT標記技術鑒定出大鼠睡眠剝奪后與對照組海馬組織相比差異表達蛋白涉及蛋白質加工與降解、突觸再生及其功能、氧化應激反應、細胞凋亡過程等等,而這些過程多數(shù)與神經系統(tǒng)退行性疾病尤其是AD發(fā)生的病理機制密切相關,提示睡眠缺失與腦老化及神經系統(tǒng)退行性疾病可能存在潛在的聯(lián)系。從而幫助我們進一步深入理解這些疾病的本質。2通過本實驗表明睡眠剝奪導致大腦海馬組織相關功能蛋白發(fā)生改變,這些蛋白參與神經系統(tǒng)退行性疾病的發(fā)生與發(fā)展,進一步表明睡眠剝奪與神經系統(tǒng)退行性疾病相關。而退行性疾病是一個慢性過程,本實驗睡眠剝奪作為一個急性應激因素,去除這一應激后即恢復睡眠后蛋白質是否會發(fā)生改變仍不十分清楚,因此睡眠剝奪與神經系統(tǒng)退行性疾病之間的關系仍須進一步研究證實。
[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.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R740

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