【作者】 谢薇；

【导师】 梁清华；

【作者基本信息】 中南大学 ， 中西医结合临床， 2007， 硕士

【摘要】 目的采用蛋白质组学技术研究胶原诱导性关节炎(collagen-induced arthritis，CIA)大鼠及不同时间点关节滑膜组织差异蛋白质表达及痹肿消汤治疗作用，探讨类风湿关节炎(Rheumatoid Arthritis，RA)的病理机制及痹肿消汤的疗效机理。方法采用牛Ⅱ型胶原和完全福氏佐剂建立CIA大鼠模型，将牛Ⅱ型胶原与完全弗氏佐剂研磨均匀后从大鼠尾根部皮内注射初次免疫，21天后再次免疫。初次免疫后2周从动物一般情况、足爪肿胀度、关节症状积分等方面评估模型，剔除复制不成功的动物。模型成功后随机分为痹肿消汤治疗组和模型对照组。同时治疗组灌服痹肿消汤(BZXD)药液，模型组灌服等量等次蒸馏水。正常对照组，不作处理，让其自由饮水。三组大鼠(正常组，模型组和BZXD治疗组)分别在造模后25天和45天处死。采用一步抽提法抽提滑膜蛋白质，运用固相pH梯度双向凝胶电泳分离各组大鼠滑膜组织的总蛋白质，用PDquest软件分析图谱，比较差异蛋白质，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)得到相应肽质指纹图谱，用Mascot查询软件搜索数据库，鉴定部分差异蛋白质。结果成功复制了CIA大鼠模型。获得分辨率较高、重复性较好的大鼠滑膜组织双向电泳凝胶图谱。正常组，25天，45天模型组，及25天，45天BZXD治疗组大鼠滑膜组织凝胶图谱的平均蛋白质点数分别为1018±37，1019±28，1008±34，1012±41，1015±35。经过质谱分析，鉴定了19个差异蛋白质，分别与细胞代谢，信号转导及细胞骨架等功能相关。结论1．初步建立了CIA大鼠模型，25天，45天滑膜组织的双向凝胶电泳图谱。获得了25天，45天差异表达的蛋白质，提示RA发病不同阶段具有不同的差异蛋白质表达。2．痹肿消汤可调节CIA大鼠不同时间点的多种蛋白质表达，提示该药对RA发病具有多靶点的治疗作用。更多还原

【Abstract】 OBJECTIVE This study aims to establish collagen-induced arthritis(CIA) rat model similar to RA pathogenesis, and compare different proteomic analysis of rats synovium among normal group, 25 days, 45 days CIA model group and paired Bizhongxiao Decoction (BZXD) treated group. It helps us identify proteins related to CIA rat pathological synovium, better understand pathogenesis of RA, as well as explore the molecular mechanism of Bizhongxiao Decoction (BZXD) which can be an effective therapy to CIA rats.METHODS CIA rat model was established. Bovine typeⅡcollagen (BIIC) and complete Freud adjuvant (CFA) were injected into the end of rats during the first immunization. They were injected second time after 21 days. Two weeks after first immunization, animals were evaluated according to their general condition, swelling of limbs and arthritis index (AI). Unsuccessfully established Animals were excluded. They were divided into Bizhongxiao Decoction (BZXD) treated group and model group randomly. BZXD treated group rats were administrated with BZX decoction, while model group rats were administrated with double distilled water. Normal group rats drink water freely. Synovial proteins were extracted, and two dimensional electrophoresis (2-DE) was used to separate total proteins from rat synovium of normal, 25 days, 45 days model and paired BZXD treated groups. Having been stained, proteins were analyzed by PDquest sottware. Some selected 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.RESULTS CIA rat model was established successfully. 2-DE patterns with high resolution and reproducibility from rat synovial proteins was obtained. The average spots of rat synovial proteins from normal, 25 day model, 45 day model and paired BZXD treated groups were 1018±37, 1019±28, 1008±34, 1012±41, 1015±35. 19. Protein spots expressing differently were identified by PMF. These proteins are related to cell metabolism, signal transduction and so forth.CONCLUSION 1. We established CIA rat model and 2-DE protein profile of normal, 25 days, 45 days and paired BZXD group rat synovium. Proteins which express differently in 25 days and 45 days rat groups were obtained. This indicates that different expression of proteins exists in different stages in process of RA.2. Bizhongxiao decoction regulates multiple protein expression in different time point of CIA rat model. It is indicated that this decoction can be a therapy with various effects.更多还原