發(fā)布時(shí)間：2018-01-01 13:25

  本文關(guān)鍵詞：基于蛋白質(zhì)組學(xué)技術(shù)對(duì)睡眠剝奪大鼠血清的初步研究　出處：《河北北方學(xué)院》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： Wistar大鼠 睡眠剝奪 學(xué)習(xí)記憶 血清 高豐度蛋白 二維電泳

【摘要】：睡眠是人體最基本、不可或缺的生理活動(dòng),是從事其它活動(dòng)的前提和基礎(chǔ)。無論是生理、還是心理上,人體都無法擺脫睡眠而長(zhǎng)期存在,睡眠質(zhì)量直接關(guān)系到人體的身心健康。當(dāng)代社會(huì),由于高節(jié)奏、高速度和信息化發(fā)展模式帶來一系列生活方式的變化,加之各種主客觀原因如疾病、噪聲等,使得睡眠剝奪的發(fā)生成為常態(tài),正日益成為醫(yī)學(xué)研究的熱點(diǎn)之一。睡眠剝奪帶來神經(jīng)系統(tǒng)、消化系統(tǒng)、心血管系統(tǒng)和免疫系統(tǒng)等多方面的不良后果,對(duì)神經(jīng)系統(tǒng)的損傷尤為明顯,表現(xiàn)為學(xué)習(xí)記憶能力降低、注意力下降及引起情緒和行為改變等。然而,睡眠剝奪引起機(jī)體損傷的確切原因和發(fā)生機(jī)理目前尚不清楚,深入研究睡眠剝奪相關(guān)分子并搜尋治療靶標(biāo),對(duì)發(fā)現(xiàn)特異性治療方法和調(diào)控藥物均十分必要。有多種建模方法用于睡眠剝奪研究,如水環(huán)境法、藥物刺激、輕柔刺激和強(qiáng)迫運(yùn)動(dòng)等,其中以水環(huán)境法最為經(jīng)典。由于水環(huán)境法模型動(dòng)物能觀察到動(dòng)物體重減輕、學(xué)習(xí)記憶能力下降和免疫力降低等現(xiàn)象,且異相睡眠時(shí)間減少、覺醒時(shí)間增加,而異相睡眠時(shí)間在恢復(fù)睡眠后顯著增加,覺醒時(shí)間則顯著減少。因此,該方法系建立REM睡眠剝奪動(dòng)物的較理想方法。本課題采用改良多平臺(tái)水環(huán)境法(Modified multiple platform method,MMPM),選取大鼠,通過研究建立了24h快速眼動(dòng)睡眠剝奪(Rapid eye movement sleep deprivation,REM SD)模型。血清蛋白譜的改變是許多疾病最直接的后果之一。血清蛋白質(zhì)組學(xué)(Serum proteomics)在研究疾病病理生理機(jī)制、藥物作用靶點(diǎn)及早期診斷特異性標(biāo)志物的發(fā)現(xiàn)等方面具有重要作用。本課題即利用所建立的睡眠剝奪模型,研究其血清蛋白質(zhì)組學(xué)的變化情況。血清蛋白組成復(fù)雜且比例懸殊,致使低豐度蛋白往往被高豐度蛋白掩蓋,導(dǎo)致其分辨率降低。因此,對(duì)血清樣本進(jìn)行處理并去除高豐度蛋白以提高低豐度蛋白的比例,是進(jìn)行相關(guān)研究最首要面對(duì)的難題。本研究首先系統(tǒng)考察了去除Wistar大鼠血清中高豐度蛋白的影響因素,優(yōu)化并改良了有機(jī)溶劑沉淀法。在上述工作的基礎(chǔ)上,最后應(yīng)用血清比較蛋白質(zhì)組學(xué)的方法,對(duì)快速眼動(dòng)睡眠剝奪模型動(dòng)物的血清蛋白表達(dá)變化情況進(jìn)行了研究。本實(shí)驗(yàn)總體步驟及結(jié)果為:(1)大鼠一般情況:睡眠剝奪組大鼠毛發(fā)光澤下降,精神稍差,較亢奮�？瞻讓�(duì)照組和環(huán)境對(duì)照組大鼠精神狀態(tài)良好、毛色整潔、光亮。24h睡眠剝奪對(duì)大鼠體重和學(xué)習(xí)記憶能力無影響。(2)wistar大鼠血清蛋白組學(xué)研究方法的建立與優(yōu)化:調(diào)研分析有機(jī)溶劑沉淀法可能影響高豐度蛋白去除效果的關(guān)鍵因素,設(shè)計(jì)并考察不同實(shí)驗(yàn)條件對(duì)血清高豐度蛋白去除的影響。建立最優(yōu)方法為以三氯乙酸:丙酮為1:9(v/v),且混合液體積為處理血清體積的4倍時(shí)去除高豐度蛋白效果最佳;洗滌血清蛋白沉淀時(shí),發(fā)現(xiàn)初始血清:丙酮為1:75(v/v)、洗滌3次時(shí)效果最佳。優(yōu)化等電聚焦過程中蛋白上樣量及等電聚焦電壓程序,建立最優(yōu)條件為上樣量1mg/膠條,且等電聚焦程序設(shè)置為50v條件下水化16h,250v線性升壓0.5h,1000v快速升壓1h,10000v線性升壓5h,10000v快速升壓至70000vh,1000v快速降壓36h。(3)差異蛋白分析:蛋白質(zhì)點(diǎn)主要分布在分子質(zhì)量20-95kda、等電點(diǎn)4.5-6.5的區(qū)域內(nèi)�？瞻讓�(duì)照組、環(huán)境對(duì)照組和睡眠剝奪組凝膠的蛋白點(diǎn)計(jì)數(shù)、與主膠匹配的蛋白質(zhì)點(diǎn)計(jì)數(shù)、匹配的蛋白質(zhì)點(diǎn)數(shù)占所在凝膠蛋白質(zhì)點(diǎn)數(shù)的百分比和匹配的蛋白質(zhì)點(diǎn)數(shù)占主膠中蛋白質(zhì)點(diǎn)數(shù)百分比均無顯著差異。在分子量約10-30kda、等電點(diǎn)5.5-6范圍內(nèi),與環(huán)境對(duì)照組和空白對(duì)照組相比,睡眠剝奪組至少有ssp號(hào)為5003、5101共2個(gè)蛋白質(zhì)點(diǎn)均表達(dá)下降。與空白對(duì)照組相比,點(diǎn)5003下降5.32倍,點(diǎn)5101下降4.41倍;與環(huán)境對(duì)照組相比點(diǎn),點(diǎn)5003下降3.86倍,點(diǎn)5101下降4.99倍。在分子量約30-50kda、等電點(diǎn)5.5-6范圍內(nèi),與環(huán)境對(duì)照組和空白對(duì)照組相比,睡眠剝奪組至少有ssp號(hào)為4310和4314共2個(gè)蛋白質(zhì)點(diǎn)均表達(dá)下降,與空白對(duì)照組相比,點(diǎn)4310下降3.38倍,點(diǎn)4314下降5.21倍;與環(huán)境對(duì)照組相比點(diǎn),點(diǎn)4310下降5.17倍,點(diǎn)4314下降4.82倍。在分子量約30-50kda、等電點(diǎn)6-6.5范圍內(nèi),與環(huán)境對(duì)照組和空白對(duì)照組相比,睡眠剝奪組至少有ssp號(hào)為6206和7201共2個(gè)蛋白質(zhì)點(diǎn)均表達(dá)下降,與空白對(duì)照組相比,點(diǎn)6206下降4.95倍,點(diǎn)7201下降3.41倍;與環(huán)境對(duì)照組相比點(diǎn),點(diǎn)6206下降4.37倍,點(diǎn)7201下降3.53倍。在分子量約50-80KDa、等電點(diǎn)5.5-6范圍內(nèi),與環(huán)境對(duì)照組和空白對(duì)照組相比,睡眠剝奪組有至少有SSP號(hào)為3606和2606共2個(gè)蛋白質(zhì)點(diǎn)均表達(dá)上升,與空白對(duì)照組相比,點(diǎn)3606上升2.18倍,點(diǎn)2606上升4.74倍;與環(huán)境對(duì)照組相比點(diǎn),點(diǎn)3606上升2.72,點(diǎn)2606上升2.69倍。(4)差異蛋白質(zhì)譜鑒定及生物信息學(xué)分析:將上述6個(gè)蛋白質(zhì)點(diǎn)SSP4310、SSP4314、SSP5003、SSP5101、SSP6206、SSP7201進(jìn)行LC-MS/MS質(zhì)譜測(cè)定和數(shù)據(jù)庫檢索,對(duì)其歸屬進(jìn)行了鑒定。結(jié)果表明,蛋白質(zhì)點(diǎn)SSP4310、SSP4314、SSP5003、SSP5101、SSP6206、SSP7201分別為Serotransferrin、Serotransferrin、Glutathione peroxidase 3、Ig kappa chain C region,B allele、Collagen alpha-2(I)chain、Collagen alpha-2(I)chain。Serotransferrin作為負(fù)性急性期反應(yīng)蛋白,其含量下降暗示機(jī)體抗氧化能力下降而導(dǎo)致氧化損傷;Glutathione peroxidase 3含量下降表明機(jī)體抗氧化能力可能下降,Ig kappa chain C region,B allele含量下降表明機(jī)體免疫力下降;Collagen alpha-2(I)chain降低可能與SD引起心臟損傷有關(guān)聯(lián)。通過上述研究,本課題建立并優(yōu)化了Wistar大鼠血清雙向電泳實(shí)驗(yàn)方法;通過對(duì)Wistar大鼠SD血清樣本的蛋白組學(xué)研究,發(fā)現(xiàn)24h睡眠剝奪后,8個(gè)蛋白發(fā)生差異表達(dá),其中顯著升高的蛋白有2個(gè),顯著下降的蛋白有6個(gè);質(zhì)譜鑒定和后續(xù)的生物信息學(xué)相關(guān)分析提示上述蛋白可能與氧化損傷、免疫力降低等病理生理功能關(guān)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.

【學(xué)位授予單位】：河北北方學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R740

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 黃建歐,趙忠新;大鼠睡眠剝奪方法的研究進(jìn)展[J];中華神經(jīng)醫(yī)學(xué)雜志;2004年03期

2 黃小玉;王忱;;突觸可塑性的結(jié)構(gòu)基礎(chǔ)和分子機(jī)制[J];重慶醫(yī)學(xué);2006年21期

3 惠雪楓;宿長(zhǎng)軍;李柱一;饒志仁;張潤(rùn)寧;;大鼠下丘腦星形膠質(zhì)細(xì)胞對(duì)不同時(shí)段睡眠剝奪的反應(yīng)[J];第四軍醫(yī)大學(xué)學(xué)報(bào);2006年06期

4 陳鋒菊;張曉羲;寧麗紅;;有機(jī)溶劑沉淀法去除子宮內(nèi)膜癌血清高豐度蛋白的比較研究[J];湖南文理學(xué)院學(xué)報(bào)(自然科學(xué)版);2013年03期

5 王勝智;王景杰;馬慶久;何顯力;南菁;秦云霞;邱建勇;;睡眠剝奪應(yīng)激對(duì)幼鼠海馬和視上核中谷氨酸受體及轉(zhuǎn)運(yùn)體表達(dá)的影響[J];醫(yī)學(xué)研究生學(xué)報(bào);2007年09期

6 馬雙雙;齊陽;王瑩;徐紀(jì)平;蔡?hào)|聯(lián);黃文;李兆申;;連續(xù)睡眠剝奪對(duì)大鼠認(rèn)知能力、神經(jīng)遞質(zhì)及海馬結(jié)構(gòu)的影響[J];海軍醫(yī)學(xué)雜志;2013年02期

7 韓俊;王彬;馬珂;閆昱博;董開源;劉芳娥;;褪黑素對(duì)睡眠剝奪大鼠胃黏膜損傷的保護(hù)作用[J];鄭州大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2013年06期

8 胡慶;楊麗軍;張山起;;微量元素鋅對(duì)睡眠剝奪大鼠學(xué)習(xí)記憶保護(hù)機(jī)制的研究[J];山西醫(yī)藥雜志(下半月刊);2008年05期

9 潘集陽;田徑;馬文彬;黃俏庭;張繼輝;Hiroshi Kurihara;;REM睡眠剝奪對(duì)大鼠焦慮行為的影響及瘦素的作用[J];實(shí)用醫(yī)學(xué)雜志;2010年13期

10 榮霏;程濱;溫曉颯;馬文領(lǐng);;慢性睡眠剝奪對(duì)大鼠學(xué)習(xí)記憶功能及不同腦區(qū)5-羥色胺1A受體蛋白表達(dá)的影響[J];上海醫(yī)學(xué);2012年05期

相關(guān)博士學(xué)位論文 前1條

1 張娜;神經(jīng)顆粒素在睡眠剝奪損害認(rèn)知功能中的作用及機(jī)制研究[D];中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院;2009年

，

本文編號(hào)：1364691