【作者】 梁清华； 杨波； 蔡颖； 谢薇； 何金华； 刘小春；

【机构】 中南大学湘雅医院中西医结合研究所；

【摘要】 目的研究痹肿消汤干预胶原诱导性关节炎大鼠滑膜病变的蛋白质改变,探讨痹肿消汤治疗RA的作用机制。方法 70只SD大鼠随机分为3组,采用牛Ⅱ型胶原与完全福氏佐剂诱导SD大鼠实验性关节炎(CIA)模型,应用双向凝胶电泳技术(two-dimensional gel electrophoresis,2DGE)分别分离正常组、CIA模型组及痹肿消汤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(Matrix-assisted laser desorption／ionization time-of-flight mass spectrometry, MALDI-TOF-MS)得到相应的肽质量指纹图谱(peptide mass fingerprint,PMF),然后搜索数据库鉴定部分差异蛋白质点。结果建立了正常组、CIA模型组和痹肿消汤组关节滑膜的双向凝胶电泳图谱,其平均蛋白质点数分别为947±39,994±41,1031±52,其匹配率为92％, 91％,94．2％;发现并鉴定了正常组、CIA模型组和痹肿消汤组大鼠滑膜的差异表达大于2 倍的蛋白质点55个,这些差异表达蛋白质的功能涉及物质代谢、能量产生、物质转运、抗氧化应激、信号转导及细胞骨架蛋白,随即用免疫印迹的方法差异蛋白质annexin Ⅰ、aldolase A在正常组、CIA模型组、痹肿消汤组中的表达,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、痹肿消汤组关节滑膜的双向凝胶电泳图谱,鉴定了55个与CIA 病变相关的差异表达的滑膜蛋白质,为揭示CIA滑膜病变的机制及RA的发病机理提供了新的线索。annexin Ⅰ、aldolase A的免疫印迹验证结果与蛋白质分析结果的一致性表明,annexin Ⅰ、aldolase A可能与CIA的关节滑膜病变有关。痹肿消汤通过对CIA大鼠滑膜蛋白质表达的调控从而达到治疗目的。更多还原

【Abstract】 Objective: To explore the proteomics study of synovitis of collagen induced arthritis (CIA) rat treated with bizhongxiao decoction (BZXD) for elucidating the treatment mechanism of BZXD. Methods: 70 rats were divided into 3 groups randomly,the experiment arthritis rat model was established by subcutaneous injection of Ⅱ collagen and complete Freunds adjuvant.The total proteins of synovial tissue of rat joint from the normal and CIA model and BZXDtreatment group were seperated by two-dimensional gel electrophoresis(2-DE), respectively. Then gels were stained by Coomassie brilliant blue, scanned by ImageScanner and analyzed with PDQuest software. Relative abundance of the protein spots between normal ang CIA model and BZXD treatment group 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 valiated some of them by western blotting. Results: The incidence of arthritis in the rats was approximately 88%.Well-resolved and reproducible 2DE maps of rat synovial tissue from normal and CIA model and BZXD treatment group were acquired. The average spots of normal and model and BZXD treatment group were 947±39, 994±41, 1031±52 respectively.the match rates were 92%, 91%. 94.2%.55 more than 2 folds differentially expressed protein spots were identified. The functions of these proteins involve in metabolism, energy generation, transportation, antioxidation, sigal transduction and protein folding etc.Then the expression difference of annexin I and annexin V in normal and CIA model and BZXD treatment group by western blotting is be similar to that of proteomic results. Conclusion: 2DE protein expression profiles of rat synovial tissue from normal and model and BZXD treatment group were established. 55 differently expressded proteins related to CIA rat synovitis were identified, which of them are possible to participate the pathogenesis process of CIA in different ways. These data may provide new and useful clues for elucidating the mechanism of synovitis of CIA and RA pathogenesis.The verified outcome of annxin Ⅰ and aldolase A by western blotting was consistent with the proteome results.It shows that annexin Ⅰ and aldolase A are related to CIA synovitis and RA. BZXD has good effect on CIA by regulating synovial tissue protein expression profiles.更多还原