【作者】 黄琳燕；

【导师】 李俊；

【作者基本信息】 安徽医科大学 ， 药理学， 2009， 硕士

【摘要】 目的:观察佐剂性关节炎(AA)大鼠外周血(PB)和淋巴结(LN)中CD4~+CD25~+调节性T细胞(CD4~+CD25~+Treg)的动态变化,并探讨其在AA发病过程中的意义;初步研究CD4~+CD25~+Treg对AA大鼠腹腔巨噬细胞(PMΦ)的可能发挥的作用,进一步阐明大鼠AA的发病机制,并为CD4~+CD25~+Treg是否能作为RA的预防与治疗的标志性指标提供一定的依据。方法和结果:1.大鼠佐剂性关节炎(adjuvant arthritis, AA)模型的制备及评价健康雄性SD大鼠,左足跖皮内注射0.1ml 10g?L-1氟氏完全佐剂(Freund’s complete adjuvant, FCA)复制AA模型;检测大鼠关节肿胀度并进行关节炎评分,HE染色法对关节组织作病理检查。结果显示:AA大鼠足爪的炎症反应强烈,关节肿胀明显;病理学检查关节组织可见大量炎性细胞浸润,滑膜细胞过度增生,滑膜组织中胶原蛋白等渗出并出现纤维沉着,提示大鼠AA模型制备成功。2. AA大鼠腹腔外周血和淋巴结CD4~+CD25~+Treg的动态变化及意义在大鼠AA模型制备成功的基础上,分别在造模后不同时间点,第1,3,10,17,21,24和28d,采集外周血和腹腔淋巴结,按文献方法制备淋巴细胞悬液,采用流式细胞术(FCM)检测CD4~+CD25~+Treg的比例变化;采用MTT法检测淋巴结淋巴细胞增殖反应;FCM检测刀豆蛋白A(ConA)刺激48h后,腹腔淋巴结和外周血淋巴细胞中CD4~+CD25~+Treg的比例变化;Pearson相关性分析淋巴结CD4~+CD25~+Treg与关节炎症状的相关程度。结果显示:CD4~+CD25~+Treg的比例在AA造模后第1,24和28d与正常组相比明显升高;AA大鼠第28d体内淋巴结淋巴细胞增殖反应减弱;ConA刺激淋巴结淋巴细胞48h后,正常的CD4~+CD25~+Treg的比例明显增加,但AA第28d的CD4~+CD25~+Treg比例没有显著性改变;AA不同时间点关节肿胀度和关节炎评分与CD4~+CD25~+Treg的比例存在正相关。3. CD4~+CD25~+调节性T细胞对大鼠AA作用的初步研究按文献方法分离腹腔淋巴细胞(LC)与腹腔巨噬细胞(PMΦ)后,将两者联合培养48h,放射免疫法检测培养上清中TNF-α和IL-6的含量。结果显示:AA 28d时,正常组LC与AA PMΦ共培养体系中TNF-α和IL-6含量稍低于模型组,但高于正常组,提示正常LC中的CD4~+CD25~+Treg对AA PMΦ有负反馈抑制作用;AA组LC+正常组PMΦ培养上清中TNF-α和IL-6含量明显低于正常组,提示AA LC中的CD4~+CD25~+Treg对正常PMΦ的功能有明显的抑制作用。结论:1.本研究发现了AA大鼠CD4~+CD25~+Treg动态变化规律,并进一步发现AA大鼠CD4~+CD25~+Treg与AA大鼠关节炎症状呈正相关。2.在腹腔LC与PMΦ的体外联合培养体系中,LC中CD4~+CD25~+Treg可能参与抑制PMΦ分泌TNF-α和IL-6。更多还原

【Abstract】 OBJECTIVE:To analyze the change of CD4~+CD25~+regulatory T cells (CD4~+CD25~+Treg) in rat adjuvant arthritis model and to investigate their preliminary roles in the progress of arthritis. To observe the possible role of CD4~+CD25~+Treg to peritoneal macrophage, elucidate the mechanism of RA and provide certain evidence whether CD4~+CD25~+Treg could be the preventive and therapeutic target for RA.METHODS AND RESULTS:1. Establishment and evaluztion of adjuvant arthritis model in ratsHealthy male SD rats were administrated with 0.1ml complete Freud adjuvant (FCA) (10g?L-1) via toe endermic injection. The change of secondary paw-swelling was observed and scored; Animals were sacrificed at every time point, Pathological changes of synovium were examined by Hematoxylin eosin (HE) stain method, then the samples of celiac lymphocyte and peritoneal macrophage were collected. The proportion of CD4~+CD25~+Treg was detected by flow cytometry. The results showed that the inflammation and paw swelling of AA rats increased significantly; The pathological examination also revealed that inflammatory cells infiltrated into the synovium, The synoviocytes were hyperplasia and collagen was exuded to form cellulose deposition. In conclusion, adjuvant arthritis model in rats was established successfully. 2. Change and its significance of CD4~+CD25~+ regulatory T cells from celiac lymph node and peripheral blood in experimental rat adjuvant arthritis modelOn the basis of successful rat adjuvant arthritis model, collectted celiac lymph nodes and peripheral blood at the different stage of AA, which were 1d, 3d, 10d, 17d, 21d, 24d and 28d, prepare lymphocyte supernants according to the relative literature; ConA- and LPS-induced splenocyte proliferation on AA rats were assayed with MTT reagent; the proportion of CD4~+CD25~+Treg as well as those stimulated with ConA for 48h was detected by flow cytometry; The relationship between CD4~+CD25~+Treg and arthritis symptom was processed by Pearson correlation analysis. The results showed that the proportion of CD4~+CD25~+Treg in AA group was significantly higher than control group at 1d, 24d and 28d; The results showed that the lymphocyte proliferation reaction of AA rats were lowered; The proportion of CD4~+CD25~+Treg in control group was significantly increased after stimulated with ConA for 48h, while the difference of AA group had no significance; There was positive correlation between CD4~+CD25~+Treg and arthritis symptom.3. Study of the preliminary role of CD4~+CD25~+Treg in AACeliac LC and PMΦwere isolated according to literature, LC was co-cultured with PMΦfor 48 hours and the cultivation supernant was collected for detecting TNF-αand IL-6 which was measured by Radioimmunoassay. The level of TNF-αand IL-6 in co-culture supernants of normal LC and AA PMΦwere slightly lower than AA PMΦ, but obviously higher than normal PMΦ, while those in co-culture supernants of AA LC and normal PMΦdecreased significantly. This suggested that CD4~+CD25~+Treg from normal LC presented a negative feedback effect on AA PMΦ, and CD4~+CD25~+Treg from AA LC exerted obviously inhibitive effect on the function of normal PMΦ.CONCLUSIONS:1. This study discovered the rule of the dynamic change of the proportion of CD4~+CD25~+ Treg from celiac lymph node and peripheral blood at different stages of AA, and the changes of CD4~+CD25~+Treg and the degree of inflammation were positively correlated.2. CD4~+CD25~+Treg may play the role in inhibting PMΦsecreting TNF-αand IL-6 in the co-culture system in vitro.更多还原