本文選題：生物化學(xué)分級(jí)分離 + 脯氨酰羥化酶beta亞基��； 參考：《吉林大學(xué)》2008年博士論文

【摘要】： 散發(fā)性帕金森�。╯poradic Parkinson' disease，sP D）等路易體疾�。↙ewybody diseases，LBD）的病變過(guò)程主要涉及三個(gè)通路，即以蛋白質(zhì)錯(cuò)誤折疊（misfolding或malfolding）為主要特征的蛋白質(zhì)聚集（protein aggregation）通路，以蛋白酶體功能障礙（dysfunction of proteasome）為主要特征的蛋白質(zhì)分解障礙(impaired protein degradation)通路，和以線粒體（mitochondrion）功能障礙為主要特征的氧化應(yīng)激（oxidative stress）通路；同時(shí)，三個(gè)通路之間通過(guò)相互重疊的支路彼此影響，從而使sPD的病理機(jī)制復(fù)雜而多樣化。其中，蛋白酶體功能障礙、蛋白質(zhì)錯(cuò)誤折疊以及蛋白質(zhì)聚集是目前有關(guān)路易（�。w[Lewy body（LB），或Lewy bodies（LBs）]形成機(jī)制的重要研究方向。在蛋白質(zhì)組分析（proteomic analysis）被應(yīng)用于揭示疾病病理機(jī)制的研究中，通過(guò)對(duì)大量蛋白質(zhì)在疾病發(fā)生過(guò)程中發(fā)生變化的整體認(rèn)識(shí)，可以獲得與疾病相關(guān)的目的蛋白質(zhì)（the protein of intrest）的具體信息。其中，含有目的蛋白質(zhì)的細(xì)胞或細(xì)胞器或蛋白質(zhì)復(fù)合體（protein complex）通過(guò)雙向凝膠電泳（twodimensionalelectrophoresis，2 Delectrophoresis）分離，質(zhì)譜（mass spectrometry，MS）分析、質(zhì)譜數(shù)據(jù)在線檢索（submission to search engine on line）和免疫組織（細(xì)胞）化學(xué)定位（localization）和（或）免疫印跡半定量分析（semiquantitative analysis）等是較為常見的蛋白質(zhì)組學(xué)研究方法。在“PC12細(xì)胞的PSI誘導(dǎo)性包含體的亞細(xì)胞蛋白質(zhì)組學(xué)”一文中，，為了從蛋白酶體持續(xù)性抑制的PD細(xì)胞模型中分離和純化蛋白質(zhì)包含體，我們通過(guò)低速離心、核酸酶降解和密度梯度離心等生物化學(xué)分級(jí)分離（biochemical processes of fractionation）方法，從人工合成蛋白酶體抑制劑（artifically synthesized proteasome inhibitor，PSI）作用48小時(shí)（hour，h）的大鼠嗜鉻細(xì)胞瘤細(xì)胞（rat pheochromocytomacell line，P C12）或副神經(jīng)節(jié)瘤細(xì)胞中制備了PSI誘導(dǎo)性包含體（PSIinducedinclusions）；為了在蛋白質(zhì)表達(dá)水平分析PSI誘導(dǎo)性包含體的蛋白質(zhì)構(gòu)成，通過(guò)雙向電泳、基質(zhì)輔助激光解析/電離-飛行時(shí)間質(zhì)譜（matrix assisted laser desorption/ionizationtime of flight MS）、和肽質(zhì)量指紋（peptide massfingerpring，PMF）檢索的蛋白質(zhì)組學(xué)方法，我們建立了PSI誘導(dǎo)性包含體蛋白質(zhì)組；為了在蛋白質(zhì)組水平進(jìn)一步地分析PSI誘導(dǎo)性包含體的構(gòu)成特征，通過(guò)對(duì)被鑒定蛋白質(zhì)所能提供的生物學(xué)信息進(jìn)行的綜合和歸納，我們關(guān)注了10個(gè)尚未報(bào)道的LBs內(nèi)的分子伴侶蛋白（chaperone proteins）和（或）分子伴侶樣蛋白（chaperonelikeproteins）；為了證明作為蛋白質(zhì)二硫鍵異構(gòu)酶家族[the protein disulfide isomerase（PDI）family]成員之一的脯氨酰羥化酶beta亞基（Prolyl4hydroxylasebeta Polypeptide，P4HB）參與了PSI誘導(dǎo)性包含體的形成過(guò)程，我們通過(guò)免疫細(xì)胞化學(xué)分析，在生物學(xué)意義上確認(rèn)了這個(gè)蛋白質(zhì)。
[Abstract]:The pathological process of the Louis body disease (Lewybody diseases, LBD), such as sporadic Parkinson's disease (sporadic Parkinson'disease, sP D), mainly involves three pathways, namely, protein aggregation (misfolding or malfolding) as the main characteristics of protein aggregation (protein aggregation) pathway and proteasome dysfunction OME) the main characteristics of the protein decomposition barrier (impaired protein degradation) pathway and the oxidative stress (oxidative stress) pathway characterized by mitochondrial (mitochondrion) dysfunction; at the same time, the three pathways interact with each other through overlapping branches, making the pathological mechanism of sPD complex and diverse. Proteasome dysfunction, protein misfolding, and protein aggregation are the important research directions for the formation mechanism of Louis (small) [Lewy body (LB), or Lewy bodies (LBs)]. In proteome analysis (proteomic analysis), the study of the pathogenesis of disease disease (proteomic analysis) has been developed by a large number of proteins in the disease. The overall understanding of the changes in the process can obtain specific information about the the protein of intrest related to the disease. Among them, the cells or organelles or protein complexes (protein complex) containing the target protein (protein complex) are separated by two-dimensional gel electrophoresis (twodimensionalelectrophoresis, 2 Delectrophoresis), mass spectrometry (mass) Spectrometry, MS) analysis, mass spectrometric data online retrieval (submission to search engine on line) and immunoblotting (localization) and / or immunoblotting semi quantitative analysis (semiquantitative analysis) are more common proteomics research methods. In the study of white matter, in order to separate and purify protein inclusions from the PD cell model of proteasome persistent inhibition, we use low speed centrifugation, nuclease degradation and density gradient centrifugation (biochemical processes of fractionation) to synthesize proteasome inhibitor (artifically) from artificial synthesis of proteasome inhibitors (artifically). Synthesized proteasome inhibitor, PSI) produced a PSI inducible inclusion body in the rat pheochromocytoma cells (rat pheochromocytomacell line, P C12) or paraganglioma cells of 48 hours (hour, H); in order to express the protein composition of the inducible inclusion in the protein expression analysis, the bi-directional electricity is used. Swimming, matrix assisted laser parsing / ionization - time of flight mass spectrometry (matrix assisted laser desorption/ionizationtime of flight MS), and proteomics methods for peptide mass fingerprinting (peptide massfingerpring, PMF), we established a PSI inducible inclusion body protein group; in order to further analyze PSI lure at the level of proteome The composition of the conductivity includes the synthesis and induction of the biological information provided by the identified proteins. We have paid attention to the 10 chaperone proteins and / or chaperonelikeproteins in the LBs, which has not been reported; in order to prove that it is the family of the protein two sulfur bond isomerase. The prolyyl hydroxylase beta subunit (Prolyl4hydroxylasebeta Polypeptide, P4HB), one of the members of the family [the protein disulfide isomerase (PDI) family], participates in the process of the formation of the PSI inducible inclusion body. We confirmed the protein in biological sense by immunocytochemical analysis.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R742.5;R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 楊艷艷;牛朝詩(shī);;泛素蛋白酶體系統(tǒng)與帕金森病[J];中華神經(jīng)醫(yī)學(xué)雜志;2006年09期

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