基于蛋白质组学技术对睡眠剥夺大鼠血清的初步研究

发布时间：2018-01-01 13:25

本文关键词：基于蛋白质组学技术对睡眠剥夺大鼠血清的初步研究　出处：《河北北方学院》2015年硕士论文　论文类型：学位论文

【摘要】：睡眠是人体最基本、不可或缺的生理活动,是从事其它活动的前提和基础。无论是生理、还是心理上,人体都无法摆脱睡眠而长期存在,睡眠质量直接关系到人体的身心健康。当代社会,由于高节奏、高速度和信息化发展模式带来一系列生活方式的变化,加之各种主客观原因如疾病、噪声等,使得睡眠剥夺的发生成为常态,正日益成为医学研究的热点之一。睡眠剥夺带来神经系统、消化系统、心血管系统和免疫系统等多方面的不良后果,对神经系统的损伤尤为明显,表现为学习记忆能力降低、注意力下降及引起情绪和行为改变等。然而,睡眠剥夺引起机体损伤的确切原因和发生机理目前尚不清楚,深入研究睡眠剥夺相关分子并搜寻治疗靶标,对发现特异性治疗方法和调控药物均十分必要。有多种建模方法用于睡眠剥夺研究,如水环境法、药物刺激、轻柔刺激和强迫运动等,其中以水环境法最为经典。由于水环境法模型动物能观察到动物体重减轻、学习记忆能力下降和免疫力降低等现象,且异相睡眠时间减少、觉醒时间增加,而异相睡眠时间在恢复睡眠后显著增加,觉醒时间则显著减少。因此,该方法系建立REM睡眠剥夺动物的较理想方法。本课题采用改良多平台水环境法(Modified multiple platform method,MMPM),选取大鼠,通过研究建立了24h快速眼动睡眠剥夺(Rapid eye movement sleep deprivation,REM SD)模型。血清蛋白谱的改变是许多疾病最直接的后果之一。血清蛋白质组学(Serum proteomics)在研究疾病病理生理机制、药物作用靶点及早期诊断特异性标志物的发现等方面具有重要作用。本课题即利用所建立的睡眠剥夺模型,研究其血清蛋白质组学的变化情况。血清蛋白组成复杂且比例悬殊,致使低丰度蛋白往往被高丰度蛋白掩盖,导致其分辨率降低。因此,对血清样本进行处理并去除高丰度蛋白以提高低丰度蛋白的比例,是进行相关研究最首要面对的难题。本研究首先系统考察了去除Wistar大鼠血清中高丰度蛋白的影响因素,优化并改良了有机溶剂沉淀法。在上述工作的基础上,最后应用血清比较蛋白质组学的方法,对快速眼动睡眠剥夺模型动物的血清蛋白表达变化情况进行了研究。本实验总体步骤及结果为:(1)大鼠一般情况:睡眠剥夺组大鼠毛发光泽下降,精神稍差,较亢奋。空白对照组和环境对照组大鼠精神状态良好、毛色整洁、光亮。24h睡眠剥夺对大鼠体重和学习记忆能力无影响。(2)wistar大鼠血清蛋白组学研究方法的建立与优化:调研分析有机溶剂沉淀法可能影响高丰度蛋白去除效果的关键因素,设计并考察不同实验条件对血清高丰度蛋白去除的影响。建立最优方法为以三氯乙酸:丙酮为1:9(v/v),且混合液体积为处理血清体积的4倍时去除高丰度蛋白效果最佳;洗涤血清蛋白沉淀时,发现初始血清:丙酮为1:75(v/v)、洗涤3次时效果最佳。优化等电聚焦过程中蛋白上样量及等电聚焦电压程序,建立最优条件为上样量1mg/胶条,且等电聚焦程序设置为50v条件下水化16h,250v线性升压0.5h,1000v快速升压1h,10000v线性升压5h,10000v快速升压至70000vh,1000v快速降压36h。(3)差异蛋白分析:蛋白质点主要分布在分子质量20-95kda、等电点4.5-6.5的区域内。空白对照组、环境对照组和睡眠剥夺组凝胶的蛋白点计数、与主胶匹配的蛋白质点计数、匹配的蛋白质点数占所在凝胶蛋白质点数的百分比和匹配的蛋白质点数占主胶中蛋白质点数百分比均无显著差异。在分子量约10-30kda、等电点5.5-6范围内,与环境对照组和空白对照组相比,睡眠剥夺组至少有ssp号为5003、5101共2个蛋白质点均表达下降。与空白对照组相比,点5003下降5.32倍,点5101下降4.41倍;与环境对照组相比点,点5003下降3.86倍,点5101下降4.99倍。在分子量约30-50kda、等电点5.5-6范围内,与环境对照组和空白对照组相比,睡眠剥夺组至少有ssp号为4310和4314共2个蛋白质点均表达下降,与空白对照组相比,点4310下降3.38倍,点4314下降5.21倍;与环境对照组相比点,点4310下降5.17倍,点4314下降4.82倍。在分子量约30-50kda、等电点6-6.5范围内,与环境对照组和空白对照组相比,睡眠剥夺组至少有ssp号为6206和7201共2个蛋白质点均表达下降,与空白对照组相比,点6206下降4.95倍,点7201下降3.41倍;与环境对照组相比点,点6206下降4.37倍,点7201下降3.53倍。在分子量约50-80KDa、等电点5.5-6范围内,与环境对照组和空白对照组相比,睡眠剥夺组有至少有SSP号为3606和2606共2个蛋白质点均表达上升,与空白对照组相比,点3606上升2.18倍,点2606上升4.74倍;与环境对照组相比点,点3606上升2.72,点2606上升2.69倍。(4)差异蛋白质谱鉴定及生物信息学分析:将上述6个蛋白质点SSP4310、SSP4314、SSP5003、SSP5101、SSP6206、SSP7201进行LC-MS/MS质谱测定和数据库检索,对其归属进行了鉴定。结果表明,蛋白质点SSP4310、SSP4314、SSP5003、SSP5101、SSP6206、SSP7201分别为Serotransferrin、Serotransferrin、Glutathione peroxidase 3、Ig kappa chain C region,B allele、Collagen alpha-2(I)chain、Collagen alpha-2(I)chain。Serotransferrin作为负性急性期反应蛋白,其含量下降暗示机体抗氧化能力下降而导致氧化损伤;Glutathione peroxidase 3含量下降表明机体抗氧化能力可能下降,Ig kappa chain C region,B allele含量下降表明机体免疫力下降;Collagen alpha-2(I)chain降低可能与SD引起心脏损伤有关联。通过上述研究,本课题建立并优化了Wistar大鼠血清双向电泳实验方法;通过对Wistar大鼠SD血清样本的蛋白组学研究,发现24h睡眠剥夺后,8个蛋白发生差异表达,其中显著升高的蛋白有2个,显著下降的蛋白有6个;质谱鉴定和后续的生物信息学相关分析提示上述蛋白可能与氧化损伤、免疫力降低等病理生理功能关系密切,值得深入研究。
[Abstract]:Sleep is the most basic human physiological activities, indispensable, is the premise and foundation to engage in other activities. Both physiological or psychological, human can not get rid of sleep and exist for a long time, sleep quality is directly related to human health. In modern society, due to the high tempo, a series of changes in lifestyle development mode high speed and information technology, and various kinds of subjective and objective factors such as disease, noise, which may become the norm of sleep deprivation, is becoming one of the hot medical research. Sleep deprivation caused the nervous system, digestive system, cardiovascular consequences and immune system in many aspects, especially the damage to the nervous system, performance to reduce the ability of learning and memory, attention and cause emotional and behavioral changes. However, the exact cause of sleep deprivation caused by body injury and its mechanism is still not Clearly, in-depth study of molecules related to sleep deprivation and search for therapeutic targets, to find specific treatment methods and control drugs are very necessary. There are a variety of modeling methods for sleep deprivation studies, such as water, drug stimulation, light stimulation and forced movement, the water environment was the most classic. Because of the water environment model the animal can observe animal weight loss, decreased learning and memory ability of immunity decrease, and paradoxical sleep time decreased, awakening time increases, depending on the phase of sleep time increased significantly during recovery after sleep, awakening time is significantly reduced. Therefore, the method of REM sleep deprivation animal using the ideal method. The modified multiple platform method (Modified multiple platform method, MMPM), selection of rats was studied by 24h REM sleep deprivation (Rapid eye movement sleep deprivat set Ion, REM, SD) model. The change of serum protein spectrum is one of the most direct consequence of many diseases. The serum proteomics (Serum proteomics) in the study of disease pathophysiology, plays an important role in drug targets and early diagnosis of specific biomarker discovery and so on. This paper is based on the sleep deprivation model study on the changes of serum proteomics. Serum protein complex composition and proportion of the poor, resulting in low abundance proteins tend to be high abundance protein cover, lead to the lower resolution. Therefore, the serum samples were processed and the removal of high abundance proteins in order to increase the proportion of low abundance proteins, is a problem of the first face. This study first investigates the factors affecting the removal of high abundance proteins in serum of Wistar rats, optimized and improved the organic solvent precipitation method on the basis of the above work, Finally application of serum comparative proteomics, the serum protein in REM sleep deprivation animal models of expression were studied. The general steps and the results of this experiment are: (1) the general condition of the rats: shiny hair falling group of sleep deprived rats, spirit is a bit poor, compared with the blank control group and the environment of excitement. The rats in the control group in good spirits, hair neat, bright.24h sleep deprivation had no effect on the body weight and the ability of learning and memory in rats. (2) to establish and optimize the research methods of Wistar rat serum proteomics: investigation and analysis of key factors of organic solvent precipitation may affect the removal of high abundance proteins, and influence of design the effects of different experimental conditions on serum high abundance protein removal. An optimal method for three chloroacetic acid: acetone 1:9 (v/v), and the volume of the mixed liquid for 4 times the volume of serum to remove high abundance proteins. The best fruit washing; serum protein precipitation, it is found that the initial serum: acetone 1:75 (v/v), washed 3 times. The best optimization of protein isoelectric focusing in the process of sample volume and isoelectric focusing voltage program, to establish the optimal conditions for sample 1mg/ strip, and the isoelectric focusing program set to 50V under the condition of 16h, 250V linear boost 0.5h, 1000V rapid 1H, 10000v linear boost 5h 10000v fast, up to 70000vh, 1000V fast buck 36h. (3): differential protein analysis protein mainly distributed in 20-95kda molecular mass, isoelectric point 4.5-6.5 in the region. The blank control group, control group and sleep deprivation environment the protein gel group point counting, counting, and the main protein glue, matching the percentage of protein spots where the gel protein spots and matching protein spots in gel protein percentage accounted for the main points were no significant differences in molecular weight. About 10-30kda, the isoelectric point range of 5.5-6, and environmental control group compared with blank control group, sleep deprivation group at least SSP number 50035101 a total of 2 protein spots were decreased. Compared with the control group, 5003 dropped 5.32 times, 4.41 times and 5101 point decline; environmental control group compared. 5003 drop 3.86 times, 5101 times. In fall 4.99 molecular weight is about 30-50kda. The isoelectric point range of 5.5-6, and environmental control group compared with blank control group, sleep deprivation group at least SSP number 4310 and 4314 a total of 2 protein spots were decreased compared with the control group, the 4310 point decline 3.38 times, 4314 decreased 5.21 times compared with the control group; environmental point, 4310 drop 5.17 times, 4314 times. In fall 4.82 molecular weight is about 30-50kda. The isoelectric point range of 6-6.5, and environmental control group compared with blank control group, sleep deprivation group at least SSP number 6206 and 7201 a total of 2 a protein The particle was decreased compared with the control group, 6206 dropped 4.95 times, 7201 decreased 3.41 times compared with the control group; environmental point, 6206 drop 4.37 times, 7201 times. In fall 3.53 molecular weight is about 50-80KDa. The isoelectric point range of 5.5-6, and the environment compared to the control group and blank control group, sleep deprivation group has at least SSP number 3606 and 2606 a total of 2 protein spots were increased, compared with the control group, 3606 increased 2.18 times, 2606 increased 4.74 times compared with the control group; the environment, 3606 rose 2.72, 2606 rise 2.69 times the difference (4). Protein identification by mass spectrometry and bioinformatics analysis: the 6 proteins SSP4310, SSP4314, SSP5003, SSP5101, SSP6206, SSP7201 were determined by LC-MS/MS mass spectrometry and database retrieval, for its ownership were identified. The results showed that the proteins SSP4310, SSP4314, SSP5003, SSP5101, SSP6206, SSP7201 and Serot respectively. Ransferrin, Serotransferrin, Glutathione, peroxidase 3, Ig kappa chain C region, B allele, Collagen alpha-2 chain (I), Collagen alpha-2 (I) chain.Serotransferrin as a negative acute phase protein, its content decreased suggesting antioxidant ability decreased and caused oxidative damage; Glutathione peroxidase 3 showed that content decreased antioxidant capacity may decline, Ig kappa chain C region, B allele decreased showed decreased immunity; Collagen alpha-2 (I) chain and SD may reduce the cause of heart injury. Through the above research, this subject set up and Wistar in rats serum electrophoresis method were optimized by the study group; protein in Wistar rats SD serum samples. Found after 24h sleep deprivation, differential expression of 8 proteins, which significantly increased the protein 2, significantly decreased the protein 6; mass spectrometry The related bioinformatics analysis suggests that the proteins mentioned above may be closely related to the pathophysiological functions such as oxidative damage and reduction of immunity. It is worthy of further study.

【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R740

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