類(lèi)風(fēng)濕關(guān)節(jié)炎患者血清及關(guān)節(jié)液中差異表達(dá)蛋白的蛋白質(zhì)組學(xué)研究
發(fā)布時(shí)間：2018-08-13 11:37
【摘要】：背景和目的 類(lèi)風(fēng)濕關(guān)節(jié)炎(RA)是一種常見(jiàn)的慢性、系統(tǒng)性自身免疫性疾病,可導(dǎo)致關(guān)節(jié)畸形和功能喪失。盡管RA的發(fā)病機(jī)制不完全清楚,但已證實(shí)自身免疫反應(yīng)在RA滑膜炎病理過(guò)程中發(fā)揮重要作用。近年來(lái)的研究表明,蛋白質(zhì)或多肽抗原的瓜氨酸化可能參與RA的發(fā)病過(guò)程。針對(duì)瓜氨酸化蛋白質(zhì)的自身抗體,如抗核周因子抗體(APF)、抗角蛋白抗體(AKA)、抗聚絲蛋白(filaggrin)抗體、抗環(huán)瓜氨酸肽抗體(ACPA)、抗瓜氨酸化修飾的波形蛋白抗體(AMCV)等,是RA特有的自身抗體。大量臨床研究表明,ACPA是早期診斷RA及判斷關(guān)節(jié)損傷的特異和靈敏的指標(biāo)。目前在RA中已發(fā)現(xiàn)抗原有II型膠原、纖維蛋白原、波形蛋白、纖維連接蛋白等,并且多以瓜氨酸化形式存在。這些抗原及其免疫復(fù)合物(IC)可以激活補(bǔ)體,刺激吞噬細(xì)胞釋放趨化因子,細(xì)胞因子,金屬蛋白酶等炎性介質(zhì),在RA關(guān)節(jié)滑膜組織炎性損傷中發(fā)揮重要作用。但是,關(guān)于瓜氨酸化蛋白等自身抗原性質(zhì)及其在RA中作用機(jī)制目前仍不明確。因此,與RA發(fā)病相關(guān)的抗原的鑒定對(duì)于進(jìn)一步了解RA的發(fā)病機(jī)制有重要意義。 本研究首先采用多重親和去除系統(tǒng)(mulitiple affinity removal system, MARS)去除人血清及關(guān)節(jié)液樣品中高豐度蛋白質(zhì),再利用二凝膠電泳(two-dimensional gel electrophoresis,2-DE)和基質(zhì)輔助激光解析電離飛行時(shí)間質(zhì)譜(matrix assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF-MS)技術(shù)研究RA患者血清和關(guān)節(jié)液差異表達(dá)的蛋白質(zhì)。在此基礎(chǔ)上,我們用同樣技術(shù)進(jìn)一步對(duì)RA患者血清差異表達(dá)的瓜氨酸化蛋白進(jìn)行了檢測(cè)研究。首先采用兔抗瓜氨酸化蛋白抗體免疫親和層析柱自RA患者和健康人血清中提取瓜氨酸化蛋白質(zhì),再通過(guò)比較蛋白質(zhì)組學(xué)技術(shù)來(lái)分析RA患者與健康對(duì)照組瓜氨酸化蛋白差異表達(dá)譜,為進(jìn)一步探討瓜氨酸化蛋白在RA的發(fā)病機(jī)制中的作用提供理論和實(shí)驗(yàn)依據(jù)。 方法 本實(shí)驗(yàn)中RA患者血清及關(guān)節(jié)液均取自2010年12月至2011年7月間在南京軍區(qū)南京總醫(yī)院就診的RA患者,健康人對(duì)照血清取自健康獻(xiàn)血者；對(duì)照組關(guān)節(jié)液來(lái)白骨性關(guān)節(jié)炎患者,無(wú)菌條件下抽取滑膜液。RA患者臨床診斷均符合2009年美國(guó)風(fēng)濕病學(xué)會(huì)(American College of Rheumatology, ACR)標(biāo)準(zhǔn)。 本研究首先采用多重親和去除系統(tǒng)(mulitiple affinity removal system, MARS)去除人血清及關(guān)節(jié)液樣品中高豐度蛋白質(zhì),再利用二維凝膠電泳(2-DE)將提取的蛋白質(zhì)進(jìn)行分離,用Image master軟件比對(duì)分析并篩選二者有表達(dá)差異的蛋白質(zhì)點(diǎn),再通過(guò)基質(zhì)輔助激光解析電離飛行時(shí)間質(zhì)譜技術(shù)(MALDI-TOF-MS)對(duì)挑選的差異蛋白進(jìn)行鑒定。用人工合成CCP免疫家兔得到兔抗CCP抗血清,用鹽析法和G蛋白親和層析法自抗血清中純化兔抗CCP抗體IgG,將兔抗CCP抗體與高流速NHs-活化瓊脂糖耦聯(lián)制作免疫親和層析柱,對(duì)RA患者血清及健康人血清中的瓜氨酸化蛋白進(jìn)行提取純化,再利用2-DE和MALDI-TOF-MS研究RA患者血清和關(guān)節(jié)液差異表達(dá)的瓜氨酸化蛋白。 結(jié)果 1、通過(guò)多重親和去除系統(tǒng)去除RA及對(duì)照組的血清和關(guān)節(jié)液中高豐度蛋白質(zhì),將提取的蛋白標(biāo)本在相同條件下進(jìn)行2-DE。結(jié)果發(fā)現(xiàn),RA患者和對(duì)照組關(guān)節(jié)液蛋白質(zhì)的2-DE圖譜分別顯示出1174、1145個(gè)蛋白質(zhì)點(diǎn),血清蛋白譜分別顯示出1649、1661個(gè)蛋白質(zhì)點(diǎn)。以1.5倍差異表達(dá)作為標(biāo)準(zhǔn),共篩選出92個(gè)關(guān)節(jié)液蛋白質(zhì)差異點(diǎn),其中在RA中有80個(gè)點(diǎn)表達(dá)上調(diào),表達(dá)顯著下調(diào)的僅有12個(gè)；34個(gè)血清蛋白質(zhì)點(diǎn)具有差異性,6個(gè)蛋白點(diǎn)在RA中表達(dá)上調(diào),28個(gè)點(diǎn)表達(dá)下調(diào)。從上述點(diǎn)中分別選出27個(gè)關(guān)節(jié)液蛋白質(zhì)點(diǎn)和11個(gè)血清蛋白質(zhì)點(diǎn)進(jìn)行質(zhì)譜鑒定。根據(jù)獲得的PMF結(jié)果,利用Mascot搜索引擎對(duì)NCBI及Swiss-Prot數(shù)據(jù)庫(kù)進(jìn)行搜索,成功鑒定出35種蛋白質(zhì),還有3種未知蛋白質(zhì)。 2、利用鹽析和G蛋白親和層析法從兔抗CCP抗血清中提取純化兔抗CCP抗體,制備出兔抗CCP抗體親和層析柱,對(duì)RA患者和健康人對(duì)照血清中的瓜氨酸化蛋白進(jìn)行提取和純化。分別取經(jīng)抗CCP抗體親合層析柱純化的RA患者與對(duì)照組血清混合蛋白,進(jìn)行2-DE電泳。結(jié)果發(fā)現(xiàn),RA患者和對(duì)照組血清瓜氨酸化蛋白的2-DE圖譜分別顯示出791和707個(gè)蛋白質(zhì)點(diǎn)。共有167個(gè)蛋白點(diǎn)符合t檢驗(yàn)和1.5倍倍數(shù)檢驗(yàn)的顯著性改變,其中有101個(gè)蛋白點(diǎn)在RA組中表達(dá)明顯上調(diào),66個(gè)蛋白點(diǎn)在RA組中表達(dá)顯著下調(diào),篩選其中差異明顯并且灰度值較高的的51個(gè)點(diǎn)進(jìn)行鑒定分析,并與Swiss-Prot數(shù)據(jù)庫(kù)進(jìn)行比對(duì),鑒定出多種與可能與RA發(fā)病相關(guān)的蛋白。 結(jié)論 本研究采用比較蛋白質(zhì)組學(xué)技術(shù)對(duì)RA患者血清及關(guān)節(jié)液中差異表達(dá)的蛋白及血清瓜氨酸化蛋白進(jìn)行了初步鑒定,這些差異性表達(dá)的蛋白涉及細(xì)胞代謝、炎癥反應(yīng)、細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)、細(xì)胞結(jié)構(gòu)蛋白等方面的功能,推測(cè)其中某些蛋白分子可能在RA的發(fā)病過(guò)程中發(fā)揮重要作用。對(duì)這些蛋白點(diǎn)進(jìn)一步鑒定,將有助于闡明RA的發(fā)病機(jī)制,為尋找RA新的診治靶點(diǎn)提供實(shí)驗(yàn)和理論依據(jù)。
[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.
【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R593.22
本文編號(hào)：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.
【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R593.22
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