發(fā)布時間：2018-07-05 17:32

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

【摘要】： 散發(fā)性帕金森�。╯poradic Parkinson' disease，sP D）等路易體疾病（Lewybody diseases，LBD）的病變過程主要涉及三個通路，即以蛋白質錯誤折疊（misfolding或malfolding）為主要特征的蛋白質聚集（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激（oxidative stress）通路；同時，三個通路之間通過相互重疊的支路彼此影響，從而使sPD的病理機制復雜而多樣化。其中，蛋白酶體功能障礙、蛋白質錯誤折疊以及蛋白質聚集是目前有關路易（小）體[Lewy body（LB），或Lewy bodies（LBs）]形成機制的重要研究方向。在蛋白質組分析（proteomic analysis）被應用于揭示疾病病理機制的研究中，通過對大量蛋白質在疾病發(fā)生過程中發(fā)生變化的整體認識，可以獲得與疾病相關的目的蛋白質（the protein of intrest）的具體信息。其中，含有目的蛋白質的細胞或細胞器或蛋白質復合體（protein complex）通過雙向凝膠電泳（twodimensionalelectrophoresis，2 Delectrophoresis）分離，質譜（mass spectrometry，MS）分析、質譜數(shù)據(jù)在線檢索（submission to search engine on line）和免疫組織（細胞）化學定位（localization）和（或）免疫印跡半定量分析（semiquantitative analysis）等是較為常見的蛋白質組學研究方法。在“PC12細胞的PSI誘導性包含體的亞細胞蛋白質組學”一文中，，為了從蛋白酶體持續(xù)性抑制的PD細胞模型中分離和純化蛋白質包含體，我們通過低速離心、核酸酶降解和密度梯度離心等生物化學分級分離（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（hour，h）的大鼠嗜鉻細胞瘤細胞（rat pheochromocytomacell line，P C12）或副神經節(jié)瘤細胞中制備了PSI誘導性包含體（PSIinducedinclusions）；為了在蛋白質表達水平分析PSI誘導性包含體的蛋白質構成，通過雙向電泳、基質輔助激光解析/電離-飛行時間質譜（matrix assisted laser desorption/ionizationtime of flight MS）、和肽質量指紋（peptide massfingerpring，PMF）檢索的蛋白質組學方法，我們建立了PSI誘導性包含體蛋白質組；為了在蛋白質組水平進一步地分析PSI誘導性包含體的構成特征，通過對被鑒定蛋白質所能提供的生物學信息進行的綜合和歸納，我們關注了10個尚未報道的LBs內的分子伴侶蛋白（chaperone proteins）和（或）分子伴侶樣蛋白（chaperonelikeproteins）；為了證明作為蛋白質二硫鍵異構酶家族[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誘導性包含體的形成過程，我們通過免疫細胞化學分析，在生物學意義上確認了這個蛋白質。
[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.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2008
【分類號】：R742.5;R329

【參考文獻】

相關期刊論文 前1條

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

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