胶原诱导性关节炎大鼠滑膜病变的蛋白质组学研究

Proteomic research on synovitis of rats with collagen-induced arthritis

目的探讨胶原诱导性关节炎(CIA)大鼠滑膜病变的蛋白质组学研究,为类风湿关节炎(RA)的发病及治疗提供新的线索。方法30只采用皮下注射牛Ⅱ型胶原与完全弗氏佐剂诱导的SD大鼠CIA诱导性关节炎(CIA)模型,应用双向凝胶电泳(2DE)技术分离正常组、CIA模型组大鼠关节滑膜的总蛋白后,经胶体考染显色、图像分析、识别差异表达的蛋白质点,应用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)得到相应的肽质量指纹图谱(PMF),然后搜索数据库鉴定部分差异蛋白质点,运用western blotting方法验证差异表达蛋白质。结果建立了正常组、CIA模型组大鼠滑膜的双向凝胶电泳图谱,平均点数为(1020±40)个蛋白质点,平均匹配率为(93．2±2．1)%；发现并鉴定了两组滑膜差异表达大于2倍的蛋白质点35个,这些与滑膜病变相关的差异表达蛋白质的功能涉及物质代谢、能量产生、物质转运、抗氧化、信号转导及细胞骨架蛋白,随即用western blotting方法验证差异蛋白质annexinⅠ、aldolase A在正常组和CIA模型组中的表达,其结果也显示了类似的表达差异。结论建立了正常组、CIA模型组大鼠关节滑膜的双向凝胶电泳图谱,鉴定了35个与CIA滑膜病变相关的差异表达的蛋白质,这些差异蛋白质可能以不同的方式参与了病变过程,为揭示CIA滑膜病变的机制及RA的发病机制提供了新的线索,而且annexinⅠ、aldolase A的验证结果与蛋白质组结果的一致性表明,AnnexinⅠ、aldolase A可能与CIA的关节滑膜病变有关。

Objective To explore the proteomic research on synovitis of rats with collagen-induced arthritis （CIA） for providing useful and new clues to elucidate the pathogenesis and treatment of rheumatoid arthritis （RA）. Methods The experimental arthritis rat model was established by subcutaneous injection of collagen tyge Ⅱ and complete freund＇s adjuvant. The total proteins of synovial tissue of rat joint from the model and the normal group were seperated by two-dimensional gel electrophoresis （2DE）, respectively. Then gels were stained by Coomassie brilliant blue, scanned by image scanner and analyzed with PDQuest software. Relative abundance of the protein spots between model and normal groups were calculated by comparing spots density volume on the gel. The differentially expressed protein spots were identified by peptide mass fingerprint （PMF） based on matrix-assisted laser desorption/ionization time of flight mass spectrometry（MALDI-TOF-MS） and database searching, then some of them were validated by western blotting. Results Well-resolved and reproducible 2DE maps of rat synovial tissue from model and normal group were acquired. The average number of protein spots per gel was 1020±40 and the match rate was （93.2±2.1）%. Thirty-five more than 2 folds differentially expressed protein spots were identified. The functions of these proteins involve in metabolism, energy generation,transportation,antioxidation, signal transduction and protein folding, etc. Then the different expression of annexin Ⅰ and aldolase A in model and normal group by Western blotting was similar to that of proteomic results. Conclusion 2DE protein expression profiles of rat synovial tissue from model and normal group are established. Thirty-five differently expressed proteins related to CIA rat synovitis are identified, which are possible to participate the pathogenesis process of RA in different ways. These data may provide useful clues for elucidating the mechanism of synovitis of CIA and RA pathogenesis. The verified result of annexin Ⅰ and aldolase A by Western blotting is related to the proteome results. It shows that annexin Ⅰ and aldolase A are related to CIA synovitis.