类风湿关节炎患者血清及关节液中差异表达蛋白的蛋白质组学研究
发布时间：2018-08-13 11:37
【摘要】：背景和目的 类风湿关节炎(RA)是一种常见的慢性、系统性自身免疫性疾病,可导致关节畸形和功能丧失。尽管RA的发病机制不完全清楚,但已证实自身免疫反应在RA滑膜炎病理过程中发挥重要作用。近年来的研究表明,蛋白质或多肽抗原的瓜氨酸化可能参与RA的发病过程。针对瓜氨酸化蛋白质的自身抗体,如抗核周因子抗体(APF)、抗角蛋白抗体(AKA)、抗聚丝蛋白(filaggrin)抗体、抗环瓜氨酸肽抗体(ACPA)、抗瓜氨酸化修饰的波形蛋白抗体(AMCV)等,是RA特有的自身抗体。大量临床研究表明,ACPA是早期诊断RA及判断关节损伤的特异和灵敏的指标。目前在RA中已发现抗原有II型胶原、纤维蛋白原、波形蛋白、纤维连接蛋白等,并且多以瓜氨酸化形式存在。这些抗原及其免疫复合物(IC)可以激活补体,刺激吞噬细胞释放趋化因子,细胞因子,金属蛋白酶等炎性介质,在RA关节滑膜组织炎性损伤中发挥重要作用。但是,关于瓜氨酸化蛋白等自身抗原性质及其在RA中作用机制目前仍不明确。因此,与RA发病相关的抗原的鉴定对于进一步了解RA的发病机制有重要意义。 本研究首先采用多重亲和去除系统(mulitiple affinity removal system, MARS)去除人血清及关节液样品中高丰度蛋白质,再利用二凝胶电泳(two-dimensional gel electrophoresis,2-DE)和基质辅助激光解析电离飞行时间质谱(matrix assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF-MS)技术研究RA患者血清和关节液差异表达的蛋白质。在此基础上,我们用同样技术进一步对RA患者血清差异表达的瓜氨酸化蛋白进行了检测研究。首先采用兔抗瓜氨酸化蛋白抗体免疫亲和层析柱自RA患者和健康人血清中提取瓜氨酸化蛋白质,再通过比较蛋白质组学技术来分析RA患者与健康对照组瓜氨酸化蛋白差异表达谱,为进一步探讨瓜氨酸化蛋白在RA的发病机制中的作用提供理论和实验依据。 方法 本实验中RA患者血清及关节液均取自2010年12月至2011年7月间在南京军区南京总医院就诊的RA患者,健康人对照血清取自健康献血者；对照组关节液来白骨性关节炎患者,无菌条件下抽取滑膜液。RA患者临床诊断均符合2009年美国风湿病学会(American College of Rheumatology, ACR)标准。 本研究首先采用多重亲和去除系统(mulitiple affinity removal system, MARS)去除人血清及关节液样品中高丰度蛋白质,再利用二维凝胶电泳(2-DE)将提取的蛋白质进行分离,用Image master软件比对分析并筛选二者有表达差异的蛋白质点,再通过基质辅助激光解析电离飞行时间质谱技术(MALDI-TOF-MS)对挑选的差异蛋白进行鉴定。用人工合成CCP免疫家兔得到兔抗CCP抗血清,用盐析法和G蛋白亲和层析法自抗血清中纯化兔抗CCP抗体IgG,将兔抗CCP抗体与高流速NHs-活化琼脂糖耦联制作免疫亲和层析柱,对RA患者血清及健康人血清中的瓜氨酸化蛋白进行提取纯化,再利用2-DE和MALDI-TOF-MS研究RA患者血清和关节液差异表达的瓜氨酸化蛋白。 结果 1、通过多重亲和去除系统去除RA及对照组的血清和关节液中高丰度蛋白质,将提取的蛋白标本在相同条件下进行2-DE。结果发现,RA患者和对照组关节液蛋白质的2-DE图谱分别显示出1174、1145个蛋白质点,血清蛋白谱分别显示出1649、1661个蛋白质点。以1.5倍差异表达作为标准,共筛选出92个关节液蛋白质差异点,其中在RA中有80个点表达上调,表达显著下调的仅有12个；34个血清蛋白质点具有差异性,6个蛋白点在RA中表达上调,28个点表达下调。从上述点中分别选出27个关节液蛋白质点和11个血清蛋白质点进行质谱鉴定。根据获得的PMF结果,利用Mascot搜索引擎对NCBI及Swiss-Prot数据库进行搜索,成功鉴定出35种蛋白质,还有3种未知蛋白质。 2、利用盐析和G蛋白亲和层析法从兔抗CCP抗血清中提取纯化兔抗CCP抗体,制备出兔抗CCP抗体亲和层析柱,对RA患者和健康人对照血清中的瓜氨酸化蛋白进行提取和纯化。分别取经抗CCP抗体亲合层析柱纯化的RA患者与对照组血清混合蛋白,进行2-DE电泳。结果发现,RA患者和对照组血清瓜氨酸化蛋白的2-DE图谱分别显示出791和707个蛋白质点。共有167个蛋白点符合t检验和1.5倍倍数检验的显著性改变,其中有101个蛋白点在RA组中表达明显上调,66个蛋白点在RA组中表达显著下调,筛选其中差异明显并且灰度值较高的的51个点进行鉴定分析,并与Swiss-Prot数据库进行比对,鉴定出多种与可能与RA发病相关的蛋白。 结论 本研究采用比较蛋白质组学技术对RA患者血清及关节液中差异表达的蛋白及血清瓜氨酸化蛋白进行了初步鉴定,这些差异性表达的蛋白涉及细胞代谢、炎症反应、细胞信号转导、细胞结构蛋白等方面的功能,推测其中某些蛋白分子可能在RA的发病过程中发挥重要作用。对这些蛋白点进一步鉴定,将有助于阐明RA的发病机制,为寻找RA新的诊治靶点提供实验和理论依据。
[Abstract]:Background and purpose
Rheumatoid arthritis (RA) is a common chronic, systemic autoimmune disease that can lead to joint deformities and loss of function. Although the pathogenesis of RA is not fully understood, it has been confirmed that autoimmune response plays an important role in the pathological process of RA synovitis. Recent studies have shown that citrullinated proteins or polypeptide antigens can be used to treat RA synovitis. Autoantibodies against citrullinated proteins, such as anti-perinuclear factor antibody (APF), anti-keratin antibody (AKA), anti-filaggrin antibody, anti-cyclic citrullinated peptide antibody (ACPA), anti-citrullinated waveform protein antibody (AMCV), are specific to RA. Antigens such as collagen type II, fibrinogen, vimentin, fibronectin, and so on have been found in RA. These antigens and their immune complexes (IC) can activate complements and stimulate phagocytes to release chemokines and cytokines. Zion, metalloproteinases and other inflammatory mediators play an important role in RA synovial tissue inflammatory injury. However, the nature of citrullinated proteins and their roles in RA are still unclear. Therefore, the identification of antigens associated with RA pathogenesis is of great significance for further understanding the pathogenesis of RA.
In this study, a multiple affinity removal system (MARS) was used to remove high abundance proteins from human serum and joint fluid samples. Two-dimensional gel electrophoresis (2-DE) and matrix assisted laser desorption/ionization mass spectrometry (MARS) were used to analyze the high abundance proteins. Ion time-of-flight mass spectrometry (MALDI-TOF-MS) technique was used to study the differentially expressed proteins in serum and joint fluid of RA patients. On this basis, we used the same technique to detect the differentially expressed citrullinated proteins in serum of RA patients. Firstly, the rabbit anti-citrullinated protein antibody immunoaffinity chromatography column was used to auto-R. Citrullinated proteins were extracted from serum of patients with RA and healthy controls. The differential expression profiles of citrullinated proteins between RA patients and healthy controls were analyzed by comparative proteomics techniques, which provided theoretical and experimental basis for further exploring the role of citrullinated proteins in the pathogenesis of RA.
Method
In this study, RA patients'serum and synovial fluid were collected from patients with RA in Nanjing General Hospital of Nanjing Military Region from December 2010 to July 2011, healthy people's serum was taken from healthy blood donors, and patients with osteoarthritis in control group were taken synovial fluid under aseptic condition. American College of Rheumatology (ACR) standard.
In this study, a multiple affinity removal system (MARS) was used to remove high abundant proteins from human serum and joint fluid samples. The proteins were separated by two-dimensional gel electrophoresis (2-DE). The differentially expressed proteins were analyzed and screened by image master software. Rabbit anti-CCP antibody IgG was purified from rabbit anti-CCP serum by salting-out method and G protein affinity chromatography. Rabbit anti-CCP antibody was coupled with high-velocity NHs-activated agarose for immunization. Citrullinated proteins were extracted and purified from RA patients'serum and healthy persons' serum by epidemic affinity chromatography. The differentially expressed citrullinated proteins in RA patients'serum and joint fluid were studied by 2-DE and MALDI-TOF-MS.
Result
1. The high-abundance proteins in serum and joint fluid of RA and control group were removed by multiple affinity removal system, and the extracted proteins were 2-DE under the same conditions. White matter dots. According to the 1.5-fold differential expression criterion, 92 different protein spots in synovial fluid were screened out, of which 80 were up-regulated and 12 were down-regulated in RA, 34 were up-regulated in serum protein spots, 6 were up-regulated in RA and 28 were down-regulated in RA. According to the PMF results, 35 proteins and 3 unknown proteins were identified by Mascot search engine in NCBI and Swiss-Prot databases.
2. Rabbit anti-CCP antibody was extracted and purified from rabbit anti-CCP antiserum by salting out and G-protein affinity chromatography. Rabbit anti-CCP antibody affinity chromatography column was prepared to extract and purify citrullinated proteins from RA patients and healthy controls. The 2-DE patterns of serum citrullinated proteins in RA patients and controls showed 791 and 707 protein spots, respectively. A total of 167 protein spots accorded with t test and 1.5 fold test. 101 protein spots were up-regulated in RA group, and 66 protein spots were down-regulated in RA group. Fifty-one points with significant difference and high gray value were screened for identification and analysis, and compared with Swiss-Prot database, a variety of proteins associated with RA pathogenesis were identified.
conclusion
In this study, comparative proteomics was used to identify differentially expressed proteins and serum citrullinated proteins in serum and joint fluid of RA patients. These differentially expressed proteins involved in cell metabolism, inflammation, cell signal transduction, cell structural proteins and other functions. It was speculated that some of these proteins might be possible. Further identification of these proteins will be helpful to elucidate the pathogenesis of RA and provide experimental and theoretical basis for finding new targets for diagnosis and treatment of RA.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R593.22
本文编号：2180868
[Abstract]:Background and purpose
Rheumatoid arthritis (RA) is a common chronic, systemic autoimmune disease that can lead to joint deformities and loss of function. Although the pathogenesis of RA is not fully understood, it has been confirmed that autoimmune response plays an important role in the pathological process of RA synovitis. Recent studies have shown that citrullinated proteins or polypeptide antigens can be used to treat RA synovitis. Autoantibodies against citrullinated proteins, such as anti-perinuclear factor antibody (APF), anti-keratin antibody (AKA), anti-filaggrin antibody, anti-cyclic citrullinated peptide antibody (ACPA), anti-citrullinated waveform protein antibody (AMCV), are specific to RA. Antigens such as collagen type II, fibrinogen, vimentin, fibronectin, and so on have been found in RA. These antigens and their immune complexes (IC) can activate complements and stimulate phagocytes to release chemokines and cytokines. Zion, metalloproteinases and other inflammatory mediators play an important role in RA synovial tissue inflammatory injury. However, the nature of citrullinated proteins and their roles in RA are still unclear. Therefore, the identification of antigens associated with RA pathogenesis is of great significance for further understanding the pathogenesis of RA.
In this study, a multiple affinity removal system (MARS) was used to remove high abundance proteins from human serum and joint fluid samples. Two-dimensional gel electrophoresis (2-DE) and matrix assisted laser desorption/ionization mass spectrometry (MARS) were used to analyze the high abundance proteins. Ion time-of-flight mass spectrometry (MALDI-TOF-MS) technique was used to study the differentially expressed proteins in serum and joint fluid of RA patients. On this basis, we used the same technique to detect the differentially expressed citrullinated proteins in serum of RA patients. Firstly, the rabbit anti-citrullinated protein antibody immunoaffinity chromatography column was used to auto-R. Citrullinated proteins were extracted from serum of patients with RA and healthy controls. The differential expression profiles of citrullinated proteins between RA patients and healthy controls were analyzed by comparative proteomics techniques, which provided theoretical and experimental basis for further exploring the role of citrullinated proteins in the pathogenesis of RA.
Method
In this study, RA patients'serum and synovial fluid were collected from patients with RA in Nanjing General Hospital of Nanjing Military Region from December 2010 to July 2011, healthy people's serum was taken from healthy blood donors, and patients with osteoarthritis in control group were taken synovial fluid under aseptic condition. American College of Rheumatology (ACR) standard.
In this study, a multiple affinity removal system (MARS) was used to remove high abundant proteins from human serum and joint fluid samples. The proteins were separated by two-dimensional gel electrophoresis (2-DE). The differentially expressed proteins were analyzed and screened by image master software. Rabbit anti-CCP antibody IgG was purified from rabbit anti-CCP serum by salting-out method and G protein affinity chromatography. Rabbit anti-CCP antibody was coupled with high-velocity NHs-activated agarose for immunization. Citrullinated proteins were extracted and purified from RA patients'serum and healthy persons' serum by epidemic affinity chromatography. The differentially expressed citrullinated proteins in RA patients'serum and joint fluid were studied by 2-DE and MALDI-TOF-MS.
Result
1. The high-abundance proteins in serum and joint fluid of RA and control group were removed by multiple affinity removal system, and the extracted proteins were 2-DE under the same conditions. White matter dots. According to the 1.5-fold differential expression criterion, 92 different protein spots in synovial fluid were screened out, of which 80 were up-regulated and 12 were down-regulated in RA, 34 were up-regulated in serum protein spots, 6 were up-regulated in RA and 28 were down-regulated in RA. According to the PMF results, 35 proteins and 3 unknown proteins were identified by Mascot search engine in NCBI and Swiss-Prot databases.
2. Rabbit anti-CCP antibody was extracted and purified from rabbit anti-CCP antiserum by salting out and G-protein affinity chromatography. Rabbit anti-CCP antibody affinity chromatography column was prepared to extract and purify citrullinated proteins from RA patients and healthy controls. The 2-DE patterns of serum citrullinated proteins in RA patients and controls showed 791 and 707 protein spots, respectively. A total of 167 protein spots accorded with t test and 1.5 fold test. 101 protein spots were up-regulated in RA group, and 66 protein spots were down-regulated in RA group. Fifty-one points with significant difference and high gray value were screened for identification and analysis, and compared with Swiss-Prot database, a variety of proteins associated with RA pathogenesis were identified.
conclusion
In this study, comparative proteomics was used to identify differentially expressed proteins and serum citrullinated proteins in serum and joint fluid of RA patients. These differentially expressed proteins involved in cell metabolism, inflammation, cell signal transduction, cell structural proteins and other functions. It was speculated that some of these proteins might be possible. Further identification of these proteins will be helpful to elucidate the pathogenesis of RA and provide experimental and theoretical basis for finding new targets for diagnosis and treatment of RA.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R593.22
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