小鼠胶原诱导性关节炎模型的建立及其免疫学变化研究

Establishment and Immunological Evaluation of Mouse Model of Collagen Induced Arthritis

目的胶原诱导性关节炎模型(collagen induced arthritis,CIA)是研究类风湿性关节炎发病机制和治疗药物筛选的理想模型,也是目前国际上公认的关节炎模型。但是,目前鲜见Ⅱ型胶原诱导CIA模型的系统免疫学变化的报道。因此,本研究采用DBA/1小鼠诱导了CIA模型,并对其免疫学改变进行了系统研究。方法将牛Ⅱ型胶原与完全弗氏佐剂混和并充分乳化,于DBA/1小鼠尾根部皮内注射进行初次免疫,20 d后同样方法进行再次免疫。应用千分尺测量CIA模型小鼠的左右两侧足掌厚度,并进行关节炎评分。酶联免疫吸附试验测定小鼠血清Ⅱ型胶原特异性抗体,Luminex技术和αLISA技术测定血清及培养上清中的细胞因子水平。结果 CIA小鼠于造模后23 d开始,陆续出现前肢、后肢的红肿、功能障碍,发病率高达100%,且随着时间的延长其关节肿胀程度呈进行性加重,关节炎评分增高。CIA小鼠脾脏指数较正常组明显升高,且Ⅱ型胶原刺激的特异性T细胞增殖明显增强。细胞因子检测结果表明,脾细胞培养上清中IFN-γ和IL-4含量及IFN-γ/IL-4比值明显升高,TNF-α和IL-1β水平亦显著升高。此外,CIA小鼠血清中存在高水平的Ⅱ型胶原特异性抗体。结论Ⅱ型胶原诱导CIA模型发病率高,免疫学改变以Th1细胞因子升高为主,兼有细胞免疫功能及体液免疫功能损伤。

Objective Collagen induced arthritis （CIA） is an ideal model for studying the mechanism of rheumatoid arthritis （RA） and exploring the anti-RA drug candidates. However, there are few studies about the systemic immune abnormality of CIA mice as yet. Thus, we established CIA model using collagen II and evaluated it. Methods Blending collagen II and Complete Freund＇s adjuvant and emulsifying them completely, then injected them to male DBA/1 mice at root of the tail. After 20 days, repeated this process. The swelling of the claw were determined using micrometer. ELISA, Luminex and ctLISA were employed to measure the collagen II-specific antibodies and multiple cytokines. Results Beginning from 23 days of injection, red swelling of the claw skin appeared in some mice. At the end of experiment, 100% mice developed arthritis symptoms. The spleen index of CIA mice increased significantly, accompanied by augmented spleen lymphocyte proliferation reaction to collagen II. At the same time, multiple cytokine levels in the supernatant of cultured spleen lymphocytes increased than control, such as IFN-γ, TNF-α and IL-1β. In addition, there was high amount collagen II-specific antibodie in the serum of CIA mice. Conelusions CIA mice characterized by abnormal activation of T cell, esoeeially Th1 cell, with a high incidence, is a suitable RA model.