胶原性关节炎大鼠血浆肿瘤坏死因子α和滑膜血管内皮生长因子表达的相关性

背景:类风湿关节炎的滑膜病变具有类肿瘤样生长的特点,表现为滑膜组织的肥厚增生,血管翳形成,对关节周围的组织产生侵蚀和破坏。在滑膜类肿瘤样病变的发展过程中有多种炎性因子和生长因子的参与,其中肿瘤坏死因子α和血管内皮生长因子对滑膜炎症的发展及滑膜血管翳的形成具有十分重要的作用。目的:同步观察胶原性关节炎大鼠不同时间点的血浆肿瘤坏死因子α含量与滑膜血管内皮生长因子表达变化,探讨肿瘤坏死因子α和血管内皮生长因子在类风湿关节炎发病机制中的作用及其相关性。设计:随机分组设计、动物实验。单位:中南大学湘雅医院中西医结合研究所。材料:实验于2003-07/11在中西医结合研究所实验室完成。选择日龄45～50d的SD大鼠40只,随机分为正常对照组10只,胶原性关节炎模型组30只。方法:采用10mg牛Ⅱ型胶原与5mL完全福氏佐剂研磨后,以每只100μL从大鼠尾根部皮内注射免疫,于21d后按上述方法和剂量再次重复免疫制作大鼠胶原性关节炎模型。依据关节红肿程度和范围及关节肿大和变形情况进行关节炎指数积分评定,关节炎指数积分越高,关节炎症状越严重。正常对照组于25d断头取血,胶原性关节炎模型组于免疫(造模)后25,30,35,40,45d分别断头取血,采用放射免疫法检测胶原性关节炎大鼠不同时间点的血浆肿瘤坏死因子α含量,同时采用免疫组织化学法检测滑膜组织血管内皮生长因子的表达水平,观察其发病时间与滑膜新生血管形成、关节炎指数积分以及肿瘤坏死因子α和血管内皮生长因子之间关系。用直线相关分析法分析肿瘤坏死因子α与血管内皮生长因子之间的关系,用等级相关分析法分析关节炎指数积分与血管内皮生长因子及肿瘤坏死因子α之间的关系。主要观察指标:胶原性关节炎发病时间与关节炎指数积分的关系,与血浆肿瘤坏死因子α含量及滑膜血管内皮生长因子表达的关系,胶原性关节炎大鼠血浆肿瘤坏死因子α与滑膜血管内皮生长因子表达相关分析。结果:纳入动物40只,均进入结果分析。随着胶原性关节炎发病时间的延长,滑膜新生血管逐渐增多、滑膜增厚、关节炎指数积分逐渐增加、肿瘤坏死因子α含量和血管内皮生长因子水平也随之升高;其关节炎指数积分与血管内皮生长因子表达水平呈正相关(r=0.535,P<0.05),与肿瘤坏死因子α含量虽有相关增高趋势,但差异无显著性(r=0.371,P>0.05)。血浆肿瘤坏死因子α含量与血管内皮生长因子表达水平呈显著正相关(r=0.893,P<0.01)。结论:肿瘤坏死因子α与血管内皮生长因子在类风湿关节炎炎症反应,滑膜新生血管形成的细胞因子网络中起重要作用,二者相互可能具有影响,相互促进,充当恶性网络循环的调控作用;是介导类风湿关节炎发生和发展以及骨质侵蚀、致残的众多因子中的关键因子。

Correlation between plasma contents of tumor necrosis factor-alpha and expressions of vascular endothelium growth factor of synovium in collagen-induced arthritis rats

BACKGROUND: Pathological change of synovium in rheumatoid arthritis (RA) has the characteristic of tumor-like growth, it appears thickening of the synovium tissue and the formatiom of pannus, which generate periarticular erosion and destruction. Multiplicate cell factors and growth factors participate in the development course of tumor-like lesion of synovium, and the tumor necrosis factor-α(TNF-α) and vascular endothelial growth factor(VEGF)play important roles in the development of RA and the formation of pannus.OBJECTIVE: To observe the contents of plasma TNF-α of collagen-induced arthritis and the expression change of VEGF of synovium at different time point, and investigate the effect and correlation of TNF-α and VEGF in the pathogenesis of RA.DESIGN: Randomized grouping experiment taking animals as subjects.SETTING: Institute integrated traditional and western medicine of Xiangya Hospital of Central South University.MATERIALS: The experiment was finished from July to November 2003 in the laboratory of institute integrated traditional and western medicine.Forty SD rats aged 45-50 days were selected, the rats were randomly divided into the normal control group (n=10) and collagen-induced arthritis model group(n=30).METHODS: 10 mg Cattle collagen type Ⅱ and 5 ml full Freund adjuvant were grinded together, 100 μL of which were intradermally injected at the root of tails of rats, collagen-induced arthritis model of rats were re-immunized with the above-mentioned methòd and dosage after 21 days. The accumulated points of arthritis index was evaluated based on degree and extent of flare and the condition of tumefaction and deformation, the higher the accumulated points of arthritis index were, the more serious the arthritis symptom was. The blood was obtained by decapitation after 25 days in normal control group, and in the collagen-induced arthritis model group the blood was obtained by decapitation 25,30,35,40,45 days after immunization (model establishing), the contents of plasma TNF-α of collagen-induced arthritis rats at different time point was detected by radio-immune assay, the level of expression of VEGF in synovium tissue were detected with immunohistochemical method simultaneously, the correlation of invasion time and the neovascularization of synovium. The accumulated points of arthritis index . TNF-α and VEGF was observed. The correlation of TNF-α and VEGF was analyzed with the linear correlation analysis, the correlation of the accumulated points of arthritis index and TNF-α. VEGF was analyzed with the rank correlation analysis.MAIN OUTCOME MEASURES: The correlation of invasion time of collagen-induced arthritis with the accumulated points of arthritis index, with the plasma content of TNF-α and the expression of VEGF, the correlation analysis of the plasma content of TNF-α of collagen- induced arthritis rats with the expression of VEGF in synovium.RESULTS: Forty rats attended the experiment, all of them entered the final analysis. the neovascularization of synovium was increased, synovium was thickening, the accumulated points of arthritis index was gradually increased, and the contents of TNF-α and level of VEGF were increased accordingly with the process of the invasion time of the collagen-induced arthritis rats; Its accumulated points of arthritis index had the positive correlation with the level of expression of VEGF (r=0.535 ,P ＜ 0.05)and had the correlated increasing tendency with the contents of TNF-α, but there was no significant difference(r=0.371 ,P ＞ 0.05 ). the plasma contents of TNF-α had the positive correlation with the level of expressions of VEGF (r=0.893,P ＜ 0.01 ).CONCLUSION: The TNF-α and VEGF have an important effect on the inflammatory reaction of RA and Cytokine network of neovascularization of synovium, they are possibly mutually influenced and promoted and have the effect of mediating the malignant network circulation; They are the key factors among multiplicate ones which mediate the generation and development of RA, bone erosion and Mutilation.