本文選題：聚乙二醇分級 + 血漿蛋白質(zhì)組學(xué)��； 參考：《重慶醫(yī)科大學(xué)》2017年博士論文

【摘要】：第一部分基于PEG分級聯(lián)合免疫親和層析的外周血漿蛋白質(zhì)組研究研究背景由于血漿成分復(fù)雜且蛋白濃度差異較大,血漿蛋白質(zhì)組學(xué)研究難度較大,特別是基于質(zhì)譜的血漿低豐度蛋白生物標(biāo)志物研究面臨巨大挑戰(zhàn)。傳統(tǒng)的單一分級方式并不能有效降低血漿的復(fù)雜程度,免疫親和層析也只能有限的提高低豐度蛋白的檢測效率。鑒于上述原因,我們通過聚乙二醇分級與免疫親和層析整合的策略進(jìn)行人血漿蛋白質(zhì)組學(xué)研究,提高血漿蛋白質(zhì)的檢測效率。方法1.通過聚乙二醇沉淀法對血漿蛋白進(jìn)行分級:依次用濃度為4%、8%、12%、16%、20%、24%和30%的聚乙二醇溶液進(jìn)行血漿蛋白質(zhì)分級。2.免疫親和層析和質(zhì)譜檢測:將分級處理后的樣品進(jìn)行免疫親和層析,并利用質(zhì)譜技術(shù)對各組分中的蛋白質(zhì)進(jìn)行檢測。3.質(zhì)譜數(shù)據(jù)分析:通過質(zhì)譜數(shù)據(jù)分析,評估聚乙二醇分級聯(lián)合免疫親和層析的方法有效性。結(jié)果1.通過凝膠染色對比不同濃度聚乙二醇分級效果,發(fā)現(xiàn)聚乙二醇濃度為4%時主要包含纖維蛋白原組分,濃度為12%時主要包含免疫球蛋白G組分,濃度為30%時主要包含白蛋白組分。2.聚乙二醇分級有效地將不同理化性質(zhì)的蛋白質(zhì)進(jìn)行了分離,BF組分蛋白質(zhì)分子量相對較高(45k Da),等電點(diǎn)范圍為5-8,CF組分等電點(diǎn)范圍4-7。3.通過質(zhì)譜分析和數(shù)據(jù)對比,發(fā)現(xiàn)聚乙二醇分級聯(lián)合免疫親和層析能夠提高血漿蛋白質(zhì)的檢測效率(約43%),低豐度蛋白質(zhì)的檢測效率提高約(65.8%)。結(jié)論聚乙二醇分級處理聯(lián)合免疫親和層析的血漿樣品處理策略可以降低血漿蛋白質(zhì)的復(fù)雜程度,提高血漿蛋白質(zhì)組學(xué)的鑒定效率。第二部分基于快速交聯(lián)和兩步沉淀法的血清白蛋白組分析研究背景在血漿蛋白質(zhì)組學(xué)研究中,我們通常會首先去除血清白蛋白等高豐度蛋白質(zhì),然而在此過程中與血清白蛋白相互作用的蛋白和肽段也會被去除。這些蛋白和肽段共同組成白蛋白組,且具有重要的病理生理作用,是潛在的疾病生物標(biāo)志物。親和吸附和化學(xué)沉淀的方法都曾被應(yīng)用于富集白蛋白組,但是這些方法還有待繼續(xù)改進(jìn)。本研究中我們利用快速交聯(lián)及聚乙二醇和乙醇兩步沉淀法富集白蛋白組,以發(fā)現(xiàn)更多的有價(jià)值的低豐度蛋白質(zhì)。方法1.甲醛交聯(lián),固定相互作用蛋白質(zhì):通過對不同濃度和不同交聯(lián)時間的血清蛋白進(jìn)行凝膠染色分析,確定甲醛交聯(lián)的最佳濃度和交聯(lián)時間。2.聚乙二醇沉淀聯(lián)合乙醇沉淀富集血清白蛋白:利用凝膠染色分析和免疫印跡分析確定富集血清白蛋白的聚乙二醇和乙醇最適濃度。3.質(zhì)譜結(jié)果生物信息學(xué)分析:與前期研究結(jié)果進(jìn)行對比,并構(gòu)建白蛋白相互作用網(wǎng)絡(luò)。結(jié)果1.通過對比確定最佳的血清交聯(lián)條件為:甲醛濃度10%,交聯(lián)時間5秒。2.濃度為12%的聚乙二醇溶液可以有效去除免疫球蛋白G對后續(xù)實(shí)驗(yàn)的影響。乙醇濃度為57%(PEG4000)和60%(PEG6000)時血清白蛋白的富集效果最好。3.質(zhì)譜分析共鑒定到171個蛋白質(zhì),其中125個蛋白質(zhì)參與構(gòu)建白蛋白相互作用網(wǎng)絡(luò),直接作用蛋白質(zhì)21個,間接作用蛋白質(zhì)104個。質(zhì)譜結(jié)果中有低豐度蛋白質(zhì)28個,其中12個為首次發(fā)現(xiàn)的白蛋白相關(guān)蛋白質(zhì)。結(jié)論基于快速交聯(lián)的聚乙二醇和乙醇兩步沉淀法可以有效富集血清白蛋白組,白蛋白組的質(zhì)譜分析發(fā)現(xiàn)新的具有臨床診斷價(jià)值的低豐度蛋白質(zhì)。第三部分大鼠中樞腦區(qū)杏仁核蛋白質(zhì)組及其在慢性溫和應(yīng)激下的變化研究背景杏仁核作為中樞神經(jīng)邊緣系統(tǒng)的一部分,可以產(chǎn)生、識別和調(diào)節(jié)情緒,被稱作“情緒腦”,在人類學(xué)習(xí)、記憶和情感等方面起重要作用,與多種神經(jīng)精神疾病緊密關(guān)聯(lián)。然而,目前此類疾病(如抑郁癥)相關(guān)的蛋白質(zhì)組學(xué)研究大多聚焦于海馬和前額葉皮層等腦區(qū),而杏仁核研究相對較少。作為抑郁情緒活動調(diào)控腦區(qū),杏仁核形態(tài)和功能變化與海馬和前額葉差異較大,其相關(guān)分子調(diào)控機(jī)制尚需探討。方法1.構(gòu)建動物模型,并進(jìn)行行為學(xué)評估:進(jìn)行為期8周的慢性溫和刺激造模,通過糖水偏好實(shí)驗(yàn)和強(qiáng)迫游泳實(shí)驗(yàn)等行為學(xué)檢測,得到對照、應(yīng)激敏感和應(yīng)激抵抗組。2.定量蛋白質(zhì)組學(xué)分析:采用基于穩(wěn)定同位素標(biāo)記的相對和絕對定量(i TRAQ)蛋白組學(xué)技術(shù)對各組杏仁核的蛋白質(zhì)表達(dá)水平變化進(jìn)行高通量質(zhì)譜檢測,數(shù)據(jù)庫檢索后進(jìn)行生物信息學(xué)分析。3.免疫印跡檢測和細(xì)胞模型構(gòu)建:選取感興趣蛋白進(jìn)行免疫印跡分析,并構(gòu)建細(xì)胞模型以探討潛在的分子調(diào)控機(jī)制。結(jié)果1.糖水偏好實(shí)驗(yàn)結(jié)果提示應(yīng)激敏感組大鼠糖水偏好程度明顯低于對照組和應(yīng)激抵抗組大鼠,強(qiáng)迫游泳實(shí)驗(yàn)結(jié)果提示應(yīng)激敏感組大鼠的不動時間明顯長于對照組和應(yīng)激抵抗組大鼠。2.質(zhì)譜檢測共定量蛋白質(zhì)2562個,其中102個蛋白質(zhì)表達(dá)存在組間差異。定位和功能分析發(fā)現(xiàn)25個(25%)突觸相關(guān)蛋白質(zhì)。3.免疫印跡分析:結(jié)果顯示NMDA和AMPA受體發(fā)生了顯著性變化,表明杏仁核中谷氨酸能傳遞受到CMS影響;調(diào)節(jié)谷氨酸能信號的PSD-95和Ca MKIIβ的表達(dá)水平在應(yīng)激敏感組中呈現(xiàn)增加。此外,涉及谷氨酸攝取的VGlu T1在兩個應(yīng)激組中也顯著增加。這些調(diào)節(jié)和協(xié)作分子的變化為應(yīng)激敏感大鼠杏仁核異常的突觸形態(tài)和功能可塑性分子機(jī)制提供了重要信息。結(jié)論該研究所發(fā)現(xiàn)的CMS敏感性相關(guān)因子(包括Glu N2A/B,PSD-95和Ca MKIIβ)可能為潛在的抗抑郁靶標(biāo);慢性溫和應(yīng)激下的杏仁核蛋白質(zhì)特別是突觸蛋白變化將有助于增進(jìn)我們對抑郁癥杏仁核突觸可塑性的理解。
[Abstract]:The first part is the research background of peripheral plasma proteome based on PEG classification combined immunoaffinity chromatography. The plasma proteomics research is difficult because of the complex plasma components and high protein concentration differences. Especially, the research on the biomarkers of plasma low abundance protein based on mass spectrometry is facing great challenges. It does not effectively reduce the complexity of plasma, and immunoaffinity chromatography can only improve the detection efficiency of low abundance proteins. In view of the above reasons, we use the strategy of polyethylene glycol classification and immuno affinity chromatography to carry out human plasma proteomics research to improve the detection efficiency of plasma protein. Method 1. through polyb Plasma protein was graded by diol precipitation method: plasma protein fractionation.2. immunoaffinity chromatography and mass spectrometry with 4%, 8%, 12%, 16%, 20%, 24% and 30% polyethylene glycol solutions in order: immuno affinity chromatography was carried out for the samples after the classification, and the.3. mass spectrometry was used to detect the number of proteins in each component by Chromatograph technique. Analysis: through mass spectrometry analysis, the effectiveness of polyethylene glycol classification combined immunoaffinity chromatography was evaluated. Results 1. by gel dyeing compared with different concentrations of polyethylene glycol classification effect, it was found that the concentration of polyethylene glycol 4% mainly contains fibrinogen component, when the concentration is 12%, the main component of the immunoglobulin G component, the concentration is 30%. The proteins of different physicochemical properties were effectively separated by.2. peg classification, the molecular weight of BF components was relatively high (45k Da), the range of isoelectric point was 5-8, and the range 4-7.3. of CF group was analyzed by mass spectrometry and data comparison. It was found that the polyethylene glycol classification combined immunoaffinity chromatography could be extracted. The detection efficiency of high plasma protein (about 43%) and the detection efficiency of low abundance proteins increased by about (65.8%). Conclusion the strategy of plasma sample treatment with PEG grading treatment combined with immuno affinity chromatography can reduce the complexity of plasma protein and improve the identification efficiency of plasma proteomics. The second part is based on rapid crosslinking and two steps. Background in the analysis of serum albumin in precipitation method, we usually remove the high abundance proteins such as serum albumin in plasma proteomics research. However, in this process, the proteins and peptides interacting with serum albumin are also removed. These proteins and peptide segments make up an albumin group and have important diseases. Physical and physiological functions are potential biomarkers of disease. Both affinity adsorption and chemical precipitation have been used to enrich albumin, but these methods remain to be improved. In this study, we have enriched white egg white groups by two step precipitation method with rapid crosslinking, polyethylene glycol and ethanol to find more valuable low abundance eggs. Method 1. formaldehyde crosslinking and immobilization of interaction protein: through gel staining analysis of serum proteins with different concentrations and different crosslinking times, the optimum concentration of formaldehyde crosslinking and crosslinking time.2. precipitation combined with ethanol precipitation and concentration of serum albumin was determined by gel staining analysis and Western blot analysis. Bioinformatics analysis of the optimal concentration of polyethylene glycol and ethanol enriched with serum albumin by.3. mass spectrometry: compared with the previous results and constructing the albumin interaction network. Results 1. through comparison, the optimal serum crosslinking conditions were determined as: the concentration of formaldehyde was 10%, and the polyethylene glycol solution with the concentration of 12% in the crosslinking time of 5 seconds was 12%. The effect of immunoglobulin G on the subsequent experiment was removed. The concentration of serum albumin was 57% (PEG4000) and 60% (PEG6000). The best effect of.3. mass spectrometry was best identified to 171 proteins, of which 125 proteins were involved in the construction of albumin interaction network, 21 direct proteins, 104 indirect proteins. There are 28 low abundance proteins in the fruit, of which 12 are the first discovered albumin related proteins. Conclusion based on the rapid crosslinked polyethylene glycol and ethanol two step precipitation method, the serum albumin group can be enriched effectively. The mass spectrometric analysis of the albumin group found the new low abundance protein with clinical diagnostic value. Third part of the central brain of rats. The amygdala proteome and its changes in chronic mild stress study background the amygdala, as part of the central nervous system, can produce, identify and regulate emotions, known as "emotional brain", play an important role in human learning, memory and emotion, and closely related to a variety of neuromental diseases. However, this is now the case. The proteomics research related to disease like disease (such as depression) is mostly focused on the hippocampus and prefrontal cortex, but the amygdala research is relatively less. As a depressive emotion regulation brain region, the amygdala morphology and function changes are different from the hippocampus and prefrontal lobe, and the related molecular regulation mechanism needs to be discussed. Method 1. the animal model is constructed. .2. quantitative proteomics analysis of stress sensitivity and stress resistance groups: comparative and absolute quantitative (I TRAQ) proteomics techniques based on stable isotopes based on stable isotopes, were performed for 8 weeks of chronic mild stimulation model, by behavioral tests such as sugar water preference and forced swimming tests. The protein expression level of the amygdala was detected by high flux mass spectrometry, and the bioinformatics analysis of.3. immunoblotting and cell model construction were carried out after the database retrieval. The interest protein was selected for Western blot analysis and the cell model was constructed to explore the potential sub regulation mechanism. Results the results of 1. sugar water preference experiment It was suggested that the degree of sugar water preference in the stress sensitive group was significantly lower than that of the control group and the stress resistance group. The forced swimming test showed that the time of the stress sensitive group was significantly longer than that of the control group and the stress resistance group. The 2562 proteins were detected by.2. mass spectrometry, of which 102 proteins were expressed in groups. Functional analysis found 25 (25%).3. immunoblotting analysis of synapse related protein: the results showed that NMDA and AMPA receptors had significant changes, indicating that the energy transfer of glutamate in the amygdala was affected by CMS; the expression level of PSD-95 and Ca MKII beta of glutamate signaling increased in the irritable sensitive group. In addition, it involved glutamate uptake. VGlu T1 also increased significantly in two stress groups. These changes and cooperating molecules provide important information for the synaptic morphology and functional plasticity of the amygdala abnormalities in stress sensitive rats. Conclusion the CMS sensitivity related factors (including Glu N2A/B, PSD-95 and Ca MKII beta) may be potential antitumor agents. The changes of protein in amygdala, especially synaptophysin in chronic mild stress, will help us to understand the synaptic plasticity of amygdala in depression.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R749.4

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