PC12细胞的PSI诱导性包含体的亚细胞蛋白质组学

发布时间：2018-07-05 17:32

本文选题：生物化学分级分离 + 脯氨酰羟化酶beta亚基　；参考：《吉林大学》2008年博士论文

【摘要】： 散发性帕金森病（sporadic Parkinson' disease，sP D）等路易体疾病（Lewybody diseases，LBD）的病变过程主要涉及三个通路，即以蛋白质错误折叠（misfolding或malfolding）为主要特征的蛋白质聚集（protein aggregation）通路，以蛋白酶体功能障碍（dysfunction of proteasome）为主要特征的蛋白质分解障碍(impaired protein degradation)通路，和以线粒体（mitochondrion）功能障碍为主要特征的氧化应激（oxidative stress）通路；同时，三个通路之间通过相互重叠的支路彼此影响，从而使sPD的病理机制复杂而多样化。其中，蛋白酶体功能障碍、蛋白质错误折叠以及蛋白质聚集是目前有关路易（小）体[Lewy body（LB），或Lewy bodies（LBs）]形成机制的重要研究方向。在蛋白质组分析（proteomic analysis）被应用于揭示疾病病理机制的研究中，通过对大量蛋白质在疾病发生过程中发生变化的整体认识，可以获得与疾病相关的目的蛋白质（the protein of intrest）的具体信息。其中，含有目的蛋白质的细胞或细胞器或蛋白质复合体（protein complex）通过双向凝胶电泳（twodimensionalelectrophoresis，2 Delectrophoresis）分离，质谱（mass spectrometry，MS）分析、质谱数据在线检索（submission to search engine on line）和免疫组织（细胞）化学定位（localization）和（或）免疫印迹半定量分析（semiquantitative analysis）等是较为常见的蛋白质组学研究方法。在“PC12细胞的PSI诱导性包含体的亚细胞蛋白质组学”一文中，，为了从蛋白酶体持续性抑制的PD细胞模型中分离和纯化蛋白质包含体，我们通过低速离心、核酸酶降解和密度梯度离心等生物化学分级分离（biochemical processes of fractionation）方法，从人工合成蛋白酶体抑制剂（artifically synthesized proteasome inhibitor，PSI）作用48小时（hour，h）的大鼠嗜铬细胞瘤细胞（rat pheochromocytomacell line，P C12）或副神经节瘤细胞中制备了PSI诱导性包含体（PSIinducedinclusions）；为了在蛋白质表达水平分析PSI诱导性包含体的蛋白质构成，通过双向电泳、基质辅助激光解析/电离-飞行时间质谱（matrix assisted laser desorption/ionizationtime of flight MS）、和肽质量指纹（peptide massfingerpring，PMF）检索的蛋白质组学方法，我们建立了PSI诱导性包含体蛋白质组；为了在蛋白质组水平进一步地分析PSI诱导性包含体的构成特征，通过对被鉴定蛋白质所能提供的生物学信息进行的综合和归纳，我们关注了10个尚未报道的LBs内的分子伴侣蛋白（chaperone proteins）和（或）分子伴侣样蛋白（chaperonelikeproteins）；为了证明作为蛋白质二硫键异构酶家族[the protein disulfide isomerase（PDI）family]成员之一的脯氨酰羟化酶beta亚基（Prolyl4hydroxylasebeta Polypeptide，P4HB）参与了PSI诱导性包含体的形成过程，我们通过免疫细胞化学分析，在生物学意义上确认了这个蛋白质。
[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.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R742.5;R329

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