类风湿关节炎动物模型中β-连环蛋白对Th17分化的影响

发布时间：2018-04-20 02:19

本文选题：类风湿关节炎 + 类风湿关节炎动物模型　；参考：《锦州医科大学》2017年硕士论文

【摘要】：目的类风湿关节炎(rheumatoid athritis,RA)是一种全身性自身免疫疾病,主要特征表现为慢性炎性关节病变,并伴全身多个系统受累。究其受多重因素的影响,发病机制目前尚不明确。随着Th17细胞亚群的发现,Th17细胞对RA的发病机制有了新的考究,同时也有研究表明Wnt/β-catenin信号也参与RA的炎正反应,具体机制仍不清楚。故本研究目的是探讨β-连环蛋白(β-catenin)在RA动物模型中对Th17细胞分化的影响。方法将8-10周的雄性C57BL/6小鼠分为三组:对照组,CIA组(类风湿关节炎模型组),CIA+β-catenin激动剂组(即注射LICL组)。疾病高峰期处死小鼠后,取小鼠血清,检测血清中的炎症因子(IL17、IL1β、IL6、IL23)。踝关节、膝关节、脾脏和淋巴结经固定石蜡包埋后用于HE染色、免疫}D化染色和免疫荧光染色,比较各组间病理改变、炎症细胞分布和蛋白分布(β-catenin、IL17和CD68)。脾脏和淋巴结中的AKT以及转录因子(TCF1、TCF4、RORγT,c-Jun)则用免疫印迹法(western blot)检测。结果1、比较CIA组和LICL组小鼠临床评分和发病率发现,LICL组评分较CIA组明显降低,发病时间较CIA组延迟,而且发病率较CIA组降低。2、HE染色显示,CIA组小鼠踝关节和膝关节的滑膜组织明显增厚,淋巴细胞、粒细胞等炎症细胞的浸润明显增加,膝关节的软骨组织可见滑膜组织中炎性细胞的浸润和侵蚀的现象,LICL组这些现象则明显好转。同时,血清中炎症因子结果显示;CIA组炎症因子显著增加,而LICL组则明显降低。3、LICL组血清中的炎症因子(IL1β,IL23,IL6,IL17)较CIA组明显降低;LICL的滑膜组织、软骨关节组织以及的IL17的浸润明显较CIA组减少4、CIA组的脾脏和淋巴结中IL17+Th细胞、CD68+巨噬细胞明显增加;而LICL处理后可以降低上两种细胞。免疫荧光双重染色结果显示,β-catenin可以表达在Th细胞和巨噬细胞。5、CIA脾脏和淋巴结中AKT、RORγT、c-Jun明显增加,而LICL组上述蛋白明显降低。CIA组脾脏中TCF1明显降低;LICL组脾脏中TCF1增加;而CIA组淋巴结中的TCF1增加,LICL组则降低。TCF4则只在LICL组的脾脏中显著增加。结论β-catenin降低是类风湿关节炎发病的重要因素。在类风湿关节中,β-catenin不仅通过抑制巨噬细胞活性而影响Th17分化,还可以直接影响其活性。在CIA的脾脏和淋巴结中,AKT可以通过影响c-Jun而促进Th17的分化。而β-catenin/TCF1在CIA的不同脏器可能通过不同机制参与Th17的分化。
[Abstract]:Objective rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammatory joint disease and multiple systemic involvement. It is affected by many factors, the pathogenesis is not clear. With the discovery of Th17 cell subsets, Th17 cells have a new understanding of the pathogenesis of RA, and some studies have also shown that Wnt/ 尾 -catenin signal is also involved in the positive response of RA, but the specific mechanism is still unclear. The aim of this study was to investigate the effect of 尾 -catenin on the differentiation of Th17 cells in RA animal model. Methods male C57BL/6 mice aged 8 to 10 weeks were divided into three groups: control group (rheumatoid arthritis model group) and 尾 -catenin agonist group (LICL group). After the mice were killed in the peak period of disease, the serum of mice was taken and the inflammatory factor IL17, IL1 尾, IL6 and IL23 were detected. The ankle joint, knee joint, spleen and lymph nodes were embedded with fixed paraffin wax for HE staining, immunological} D staining and immunofluorescence staining. The pathological changes, distribution of inflammatory cells and protein distribution (尾 -cateninine IL-17 and CD68) were compared among the three groups. The AKT in spleen and lymph nodes and the transcription factor TCF1, TCF4, ROR 纬 -Tnc-Jun) were detected by Western blot (Western blot). Results 1.Compared with the clinical score and incidence rate of CIA group and LICL group, the score of LICL group was significantly lower than that of CIA group, and the onset time was longer than that of CIA group. Compared with CIA group, the incidence of the disease was lower than that of CIA group. The results of HE staining showed that the synovial tissue of ankle and knee joint was thickened, and the infiltration of inflammatory cells such as lymphocytes and granulocytes was obviously increased in the CIA group. The infiltration and erosion of inflammatory cells in synovial tissue were observed in cartilage tissue of knee joint. At the same time, the results of inflammatory cytokines in serum showed that the inflammatory factors in the CIA group were significantly increased, while the inflammatory cytokines in the serum of the LICL group were significantly lower than those in the CIA group, and that in the LICL group was significantly lower than that in the CIA group. Compared with CIA group, the infiltration of IL17 Th cells and CD68 macrophages in IL17 Th cells in spleen and lymph nodes were significantly decreased in the chondroarticular tissue and IL17 group, but the CD68 macrophages in the IL17 Th cells in the spleen and lymph nodes were decreased after LICL treatment. The results of double immunofluorescence staining showed that 尾 -catenin could be expressed in the spleen and lymph nodes of Th cells and macrophages. The expression of AK Tor 纬 Tnc-Jun in spleen and lymph nodes was significantly increased, while the expression of TCF1 in spleen of LICL group was significantly lower than that of control group. The increase of TCF1 in spleen of LICL group was significantly lower than that of control group. However, the increase of TCF1 in lymph nodes in CIA group and the decrease of TCF4 in LICL group were only significantly increased in spleen of LICL group. Conclusion the decrease of 尾-catenin is an important factor in the pathogenesis of rheumatoid arthritis. In rheumatoid joints, 尾 -catenin not only affects Th17 differentiation by inhibiting macrophage activity, but also directly affects its activity. In the spleen and lymph nodes of CIA, AK T can promote the differentiation of Th17 by affecting c-Jun. 尾 -catenin / TCF1 may participate in the differentiation of Th17 in different organs of CIA through different mechanisms.
【学位授予单位】：锦州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R593.22;R-332

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