【作者】 高德玉；

【导师】 李晓军；

【作者基本信息】 南京大学 ， 临床检验诊断学， 2012， 硕士

【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。更多还原

【Abstract】 Background and PurposeRheumatoid arthritis (RA) is a common chronic inflammatory autoimmune disease characterized by persistent inflammation of the synovium,leading to local destruction of bone and cartilage and a variety of systemic manifestations that could lead to disability.Although the pathogenesis of RA is not fully understood, autoimmune reactions are suggested to play pathological roles in chronic synovitis.So far,The pathological nature of citrullination of protein in joints tissue has been recognized as something that can help us understand fundamental etiologic and pathogenetic features of rheumatoid arthritis.Since then,several autoantibodies against citrullinated proteins have been identified in RA.The antibodies such as anti-keratin antibodies(AKA),anti-perinucIear factor(APF), anti-filaggrin antibodies(AFA), anti-cyclic citrullinated peptide(CCP) antibodies are very specific in RA.Antibodies against citrullinated proteins (ACPAs) are considered as very specific markers for the early diagnosis of RA and the reflection of destruction of bone and cartilage.Thecitrullinated protein and formation of IC were responsible for inducing a spectrum of pro-inflammatory cytokines by peripheral blood mononuclear cells and macrophages via Fc receptors. So far, a variety of candidate autoantigens such as collagen type Ⅱ,fibrin/fibrinogen,vimentin,cartilage intermediate layer protein, and Epstein-Barr virus nuclear antigen-1have been suggested to induce cellular and/or humoral autoimmune responses in RA,all of those proteins are citrullinated in RA synovial tissue.The citrullinated protein and immune complexes of rheumatoid arthritis patients serum and synovial fluid (SF) are involved in the activation of the complement cascade in RA synovial tissue,and induce the macrophages to secret cytokines, metalloproteases,etc, which caused the damage of joints and cartilage.However,the concrete pathogenic mechanism of citrullinated protein remains unclear. As RA is an serious inflammatory autoimmune disease,identify the antigens of RA is became very important. After identification of these antigens, a better understanding of the immunological process in the affected joints can be achieved.In our research,the protein were abstracted from serum by mulitiple affinity removal system(MARS) human-14,differential expression of protein in serum and synovial fluid between the control and RA group were researched through two-dimensional gel electrophoresis (2-DE) and mass spectrometry(MS).In addition,the differential expression of citrullinated protein in serum were also researched.The protein were abstracted from serum by affinity chromatography with immobilized anti-cyclic citrullinated peptide,then identified through proteomics.The protein profiles of differential expression between the control and experimental group were established and some differential proteins were found. The data obtained in this study might be helpful to illuminate the mechanism of citrullination in rheumatoid arthritis.MethodsThe patients,who were diagnosed according to the American Rheumatism Association criteria, obtained from clinic and hospitalization patients from Jinling hospital and Gulou hospital affiliated to medical college of Nanjing university, Serum samples from age-and gender-matched SLE and healthy donors were used as a control.The plasma samples was collected by centrifugalization and stored at-70℃.In order to research differential expression of protein in serum and synovial fluid,the mulitiple affinity removal system(MARS) human-14were used to removel high-abundance protein in serum and synovial fluid,then differential expression of protein between the control and RA group were researched through two-dimensional gel electrophoresis (2-DE) and mass spectrometry(MS).In addition,the differential expression of citrullinated protein in serum were also researched.Protein from serum of RA and healthy control were purified by affinity chromatography with immobolized anti-cyclic citrullinated peptide antibodies.The protein of serum and synovial fluid from RA patients and control were separated by two-dimensional gel electrophoresis(2-DE).After staining by silver,data analysing image by Image Master, several significantly different spots were excised and digested by tryspin, then identified by MALDI-TOF-MS.The functions of proteins in serum were analyzing according to NCBI human database.ResultsAfter remove high-abundance protein in serum and synovial fluid through mulitiple affinity removal system,the protein were separated under the same condition.Identification by MALDI-TOF-MS of differentially expressed protein in in serum and synovial fluid shows that1174spots in synovial fluid of RA can be detected while only1145spots in control,and1649spots can detectedin serum of RA,compare to1661spots in control.6of the34distinguishing proteins in serum were differentially3-fold more overexpressed in RA compared with controls. Meanwhile,12of92spots identified proteins in synovial fluid showed their expressions to be dereased in RA.Identification by MALDI-TOF-MS of differentially expressed protein in in serum and synovial fluid,according to the peptide mass fingerprinting,35proteins has been successfully identified.The anti-CCP antibodies were purified by affinity chromatography with immobilized protein G,then protein from serum of RA and healthy control were purified by affinity chromatography with immobolized anti-cyclic citrullinated peptide antibodies.After separated by two-dimensional gel electrophoresis(2-DE),791spots in serumof RA can be detected while only707spots in control.65of the167distinguishing proteins were differentially3-fold more overexpressed in RA compared with control. Meanwhile,101spots identified proteins showed their expressions to be decreased in RA.The51specifically protein spots in RA were selected for identification by MALDI-TOF-MS.Swiss-Prot and NCBI hunman database were used to analyze these diverse proteins in sera and synovial fluid from patients of rheumatoid arthritis and healthy controls.These diverse proteins may play an importment role in the pathogenesis of rheumatoid arthritis. ConclusionIn our reaserch,the protein of differentially expressed protein and citrullinated peptide from serum and synovial fluid of RA and healthy control were identified through comparative proteomics.The diverse proteins are involved into metabolism,cell signal protein,cell structure proteins,molecular chaperones and so on.Identification of differentially expressed protein and citrullinated peptide could greatly contribute to understanding the pathogenesis of RA,and provide important assistance for the early diagnosis,clinical observation and treatment for RA.Identification of key proteins could prompt us into further understand of RA pathophysiology and may reveal new treatment target.更多还原