睡眠剥夺后海马蛋白质组学研究

发布时间：2018-06-19 01:41

本文选题：睡眠剥夺 + 氧化应激　；参考：《河北医科大学》2015年硕士论文

【摘要】：目的:睡眠在人类生活中占有很重要的位置。睡眠剥夺(sleep deprivation,SD)是指由于现代生活及工作上的需要、周围环境或其他无法避免的原因而不能保证正常的睡眠。睡眠剥夺对人类认知功能的影响是不容忽视的,包括清醒度、警觉性和注意力下降;学习记忆能力降低;执行功能下降;负面情绪增加等。睡眠缺失引起的这些一系列认知功能的障碍,与睡眠缺失导致脑部的某些重要的功能区域及有关其功能的蛋白质的改变密切相关。睡眠缺失后可以在海马区观察到很多变化如神经元的更换、凋亡,树突和突触的重构,在啮齿类动物的前额叶皮层及杏仁核也可以观察到其他一些适应性改变。在睡眠剥夺后海马神经元有明显损伤,神经元形态的改变直接影响其正常生理功能的发挥,睡眠剥夺可引起大鼠海马神经元凋亡发生。海马是位于脑颞叶内的一个部位,它是组成大脑边缘系统的一部分,担当着关于记忆以及空间定位的作用。海马亦是老年期痴呆患者较早发生病理改变的部位。有研究认为睡眠剥夺与很多神经退行性疾病密切相关,而多种神经退行性疾病均发现有某些致病蛋白质的聚集,由此可知,直接对睡眠剥夺后脑组织的蛋白质组变化的研究可更加直观的揭示其引起的机体各系统变化尤其是神经系统改变的病理生理机制。蛋白质是生物体基因功能活动的最终执行者,并直接体现生命现象的复杂性以及多样性。蛋白质组学技术的兴起是进入后基因组时代的标志,它是从整体的角度来研究蛋白质的特征,包括蛋白质的表达水平、翻译后修饰、蛋白与蛋白之间的作用等,并揭示蛋白质功能与细胞生命现象之间的规律。差异蛋白质组学则是分析不同生物体在不同状态下蛋白质表达的差异,为探索细胞的分子机制、疾病的病理病因及寻找治疗药物靶点提供了可能性的依据。本研究通过对大鼠睡眠剥夺模型的制作,对正常对照组海马组织和睡眠剥夺72小时后海马组织进行差异蛋白质组学研究,找出表达不同的蛋白质,从而探寻睡眠剥夺后引起一系列的蛋白质改变,从而帮助我们进一步深入理解这些蛋白质改变引起一系列疾病的本质。方法:1实验分组:成年雄性Wistar大鼠分为实验组和对照组,实验组大鼠进行72小时睡眠剥夺,对照组大鼠维持正常睡眠-觉醒周期。2制作大鼠睡眠剥夺模型,实验组大鼠于睡眠剥夺72小时后处死,断头取脑,分离大脑海马组织;对照组同样取相应部位脑组织,将组织放EP管中后置于-80℃冰箱中备用。3进行大鼠海马组织蛋白质提取并使用胰蛋白酶酶解脱盐后待用。4将制备好的样品肽段用TMT标记,上样buffer溶解,胶条水化,并经TFA酸化后进行C18 Zip-Tip脱盐。5抽干样品后溶解、离心,并用LC-LTQ-MS/MS分析以及质谱数据检索。结果:1成功制作大鼠睡眠剥夺模型,运用蛋白质组学及TMT技术,比较睡眠剥夺72小时组与正常对照组海马蛋白质的变化,得出差异蛋白99个,其中表达上调的蛋白有65个,表达下调的蛋白有34个。2运用Gene Codis软件分析发现有意义的蛋白60多个,这些蛋白主要涉及到蛋白质转运及降解、突触再生、氧化应激、细胞周期、能量代谢与细胞凋亡等生物学过程。结论:1运用蛋白质组学及TMT标记技术鉴定出大鼠睡眠剥夺后与对照组海马组织相比差异表达蛋白涉及蛋白质加工与降解、突触再生及其功能、氧化应激反应、细胞凋亡过程等等,而这些过程多数与神经系统退行性疾病尤其是AD发生的病理机制密切相关,提示睡眠缺失与脑老化及神经系统退行性疾病可能存在潜在的联系。从而帮助我们进一步深入理解这些疾病的本质。2通过本实验表明睡眠剥夺导致大脑海马组织相关功能蛋白发生改变,这些蛋白参与神经系统退行性疾病的发生与发展,进一步表明睡眠剥夺与神经系统退行性疾病相关。而退行性疾病是一个慢性过程,本实验睡眠剥夺作为一个急性应激因素,去除这一应激后即恢复睡眠后蛋白质是否会发生改变仍不十分清楚,因此睡眠剥夺与神经系统退行性疾病之间的关系仍须进一步研究证实。
[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.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R740

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