【作者】 王婷玉；

【导师】 李俊；

【作者基本信息】 安徽医科大学 ， 药理学， 2009， 博士

【摘要】 类风湿性关节炎(rheumatoid arthritis, RA)是一种以关节滑膜炎为特征的慢性、全身性、自身免疫性疾病,其病因及发病机制目前仍不清楚。近年来,人们发现CD4+CD25+调节性T细胞(CD4+CD25+ regulatory T cells, CD4+CD25+Tregs)是一群具有独特作用方式和功能特征的T细胞亚群,具有免疫调节作用。其在RA中的作用受到人们的广泛关注,有望成为治疗RA和筛选抗RA药物的靶点之一。大量研究表明,CD4+CD25+Tregs能够抑制自身反应性T细胞的活化与效应功能,以“主动”方式维持免疫耐受和自身稳定。越来越多的证据表明,RA患者体内CD4+CD25+Tregs存在异常,CD4+CD25+Tregs的数量和/或功能不足可能与RA的发生、发展有关。CD4+CD25+Tregs在自身免疫性疾病中的表达已成为目前研究的热点,但有关CD4+CD25+Tregs在RA中的表达与作用机制尚不明确,值得进一步深入研究。因此,本课题选用大鼠胶原性关节炎(collagen-induced arthritis, CIA)这种国际公认的RA动物模型,在成功提取纯化CⅡ、建立大鼠CIA模型的基础上,动态监测外周血中CD4+CD25+Tregs的比例变化,分析其在关节炎病程中的作用。并且以CD4+CD25+Tregs的主要细胞因子TGF-β1为切入点,探讨CD4+CD25+Tregs对腹腔巨噬细胞的调控机制及抗关节炎药来氟米特的作用,为研究RA的发病机制和治疗提供新的理论依据。研究内容主要包括以下三个部分:1.提取和纯化鸡Ⅱ型胶原,建立大鼠胶原性关节炎模型采用酸性条件下蛋白酶解与中性条件下层析洗脱的双重方法,成功获得乙酸可溶性鸡Ⅱ型胶原蛋白(Collagen TypeⅡ, CⅡ),得率约为0.17%。经SDS-PAGE和全波长紫外扫描检测,确定其与Sigma公司CⅡ标准品一致。采用CⅡ建立大鼠胶原性关节炎(Collagen-induced arthritis,CIA)模型,从体重、足爪肿胀度、多发性关节炎评分、血清中抗CⅡ抗体浓度、病理等几个方面评价,确定模型制备成功。经4次反复实验,造模成功率约为42%。2.研究CIA大鼠CD4+CD25+调节性T细胞对腹腔巨噬细胞的调控机制动态监测大鼠CIA病变过程中外周血CD4+CD25+Foxp3+T细胞的比例,与足爪肿胀做相关性分析。结果表明:CIA大鼠足爪肿胀与CD4+CD25+调节性T细胞(CD4+CD25+Tregs)的比例呈负相关,相关系数为-0.786。免疫荧光和RT-PCR结果显示,CIA大鼠CD4+CD25+调节性T细胞(CD4+CD25+Tregs)和腹腔巨噬细胞(Peritoneal macrophage,PMΦ)上分别表达TGF-β和TGF-βRII(Tβ-RII),说明TGF-β是CD4+CD25+Tregs的重要细胞因子,CD4+CD25+Tregs可通过TGF-β对巨噬细胞(macrophage, MΦ)起作用,与文献报道的结果相一致。采用TGF-β与CIA大鼠PMΦ体外共培养,研究TGF-β对CIA大鼠PMΦ免疫功能的影响及作用机制。结果表明:TGF-β(5ng/ml, 36h)可抑制LPS刺激的CIA大鼠PMΦ炎性细胞因子TNF-α和IL-6的分泌及mRNA的表达;促进抑炎性细胞因子IL-10的分泌和mRNA的表达;抑制NO的分泌及iNOSmRNA的表达。RT-PCR、流式细胞术及Western blot结果表明,TGF-β(5ng/ml, 36h)可抑制LPS刺激的CIA大鼠PMΦTLR4 mRNA和蛋白的表达,以上结果表明TGF-β可通过LPS-TLR4通路影响CIA大鼠PMΦ的免疫功能。3.探讨来氟米特对CIA大鼠CD4+CD25+调节性T细胞调控腹腔巨噬细胞的影响首先,来氟米特可以直接对CIA大鼠CD4+CD25+Tregs产生影响。体内实验结果表明,来氟米特(Lef 2 mg/kg,d12-d33)对CIA大鼠具有治疗作用,可升高CIA大鼠脾淋巴细胞中CD4+CD25+Tregs的比例及Foxp3 mRNA的表达。体外ConA诱导的CIA大鼠脾淋巴细胞增殖体系中,A771726(0.1、1、10μM)可促进脾淋巴细胞向CD4+CD25+Tregs分化,增加TGF-β的分泌,从而抑制ConA介导的脾淋巴细胞增殖活性。其次,来氟米特可以直接抑制CIA大鼠腹腔巨噬细胞的功能。体外实验结果表明,A771726(0.1、1、10μM)通过LPS-TLR4通路抑制CIA大鼠PMΦ的分泌功能。第三,来氟米特可以加强CD4+CD25+Tregs对PMΦ的调控作用。具体表现为:体外实验中,A771726(0.1、1、10μM)可加强TGF-β对CIA大鼠PMΦ分泌炎性细胞因子的抑制作用、促进抑炎性细胞因子的分泌;加强TGF-β对PMΦ产生NO的抑制作用。RT-PCR及Western blot结果表明,A771726可通过进一步抑制CIA大鼠PMΦTLR4的表达,从而加强TGF-β对PMΦ的调控作用。结论:①制备CⅡ建立大鼠CIA模型,在临床表现、免疫学和组织病理学方面均与人类RA类似,提示模型制备成功。②CIA大鼠CD4+CD25+Tregs对PMΦ的调控机制:CD4+CD25+Tregs可通过TGF-β与PMΦ上的Tβ-RII结合,抑制PMΦ表面TLR4的表达,从而抑制LPS刺激的CIA大鼠PMΦ的免疫功能。③来氟米特对CIA大鼠具有治疗作用,其机制可能有三:提高CIA大鼠抑制性细胞CD4+CD25+Tregs的比例;直接抑制PMΦ的免疫功能;通过LPS-TLR4途径加强CD4+CD25+Tregs对PMΦ的调控作用。更多还原

【Abstract】 Rheumatoid arthritis is a chronic, systemic and self-immunological disease, whose origin and pathogenesis is still unknown. In recent years, CD4+CD25+ regulatory T cells (CD4+CD25+Tregs) that possess unique functions on regulating immunity have been founnd. Its role in RA have been emphasized a lot which show that it could be a new target point of anti-arthritic drugs.Many studies demonstrated that CD4+CD25+Tregs could inhibit the activity and function of autoreactive T cells, which maintain immunologic tolerance and homoiostasis in a positive way. The abnormality of CD4+CD25+Tregs in quantity and function related to the development of autoimmune disease. More and more evidence indicate that CD4+CD25+Tregs are abnormal in RA patients and its disability is related to the occurrence and development of RA. The expression of CD4+CD25+Tregs in autoimmune disease has been focused on, but the expression and mechanism of CD4+CD25+Tregs in RA is still not well-known and need further studying. In this study, collagen-induced arthritis (CIA) in rats which well represents RA is chosen, based on abstracting and purification of collagen type II. The kinetics of CD4+CD25+Tregs population was determined to analyze its effect in CIA course. The main cytokine TGF-β1 was chose to investigate the mechanism of regulation of CD4+CD25+Tregs on peritoneal macrophages in CIA rats and the effects of anti-arthritic drug leflunomide on the regulation, to clarify the mechanism of RA and to find new pathways in cure RA. The study contains three sections: 1. Extract and purify collagen type II, and induce collagen-induced arthritis in rats.Soluble collagen typeⅡ(CⅡ) was released from the chicken sternums with pepsin digestion and passed over a DE-52 column. Soluble collagen typeⅡ(CⅡ) was released from the chicken sternums with pepsin digestion and passed over a DE-52 column. The yield of CⅡis 0.17%. SDS-PAGE and Ultra-violet scanning results of the sample and standard CⅡfrom Sigma Company were the same. Collagen-induced arthritis in rats was induced with this product, and the animal model was successful by determing body weight, paw swelling, polyarthritis index, anti-CⅡantibody in serum and histopathology examination. The achievement ratio of 4 experiments is 42%.2. Study the mechanism of regulation of CD4+CD25+ regulatory T cells on peritoneal macrophages in CIA rats.Detect the kinetics of CD4+CD25+Foxp3+ T cell ratio during the course of CIA in peripheral blood of rats, and analyze it with paw swelling. The results indicated that the paw swelling is negatively related to the percentage of CD4+CD25+Tregs in CIA rats, and the coefficient correlation is -0.786. The results in immunofluorescence and RT-PCR showed that CD4+CD25+Tregs and peritoneal macrophages (PMΦ) in CIA rats express TGF-βand Tβ-RII, respectively. This indicated that CD4+CD25+Tregs could effect on macrophages through TGF-β, which is in accordance with the literature.TGF-βwas incubated with PMΦfrom CIA rats in vitro to study the effects and mechanism of it on the immune function of CIA rats. The results showed that TGF-β(5 ng/ml, 36h) could suppress the secretion and mRNA expression of TNF-αand IL-6, improve the secretion and mRNA expression of anti-inflammatory IL-10, and inhibit the secretion of NO and mRNA expression of iNOS in PMΦfrom CIA rats. The results of RT-PCR, flow cytometry and Western blot indicated that TGF-β(5 ng/ml, 36h) inhibits mRNA and protein expression of TLR 4 in PMΦof CIA rats, which suggests that TGF-βcould regulate the immune function of PMΦfrom CIA rats through LPS-TLR4 pathway.3. Investigate the effects of leflunomide on the regulation of CD4+CD25+Tregs on PMΦof CIA rats.Firstly, leflunomide has effects on CD4+CD25+Tregs of CIA rats. The results in vivo experiment showed that leflunomide (2 mg/kg, d12-d33) has therapeutic effect on CIA rats, could increase the percentage of CD4+CD25+T cell and the expression of Foxp 3 mRNA. In in vitro Con A-induced splenic lymphocytes proliferation system, A771726 (0.1, 1, 10μM) promoted lymphocytes differentiation to CD4+CD25+Tregs and increased the secretion of TGF-β, which results in inhibiting the proliferation in Con A-stimulated splenic lymphocytes system.Secondly, leflunomide directly inhibited the immune function of PMΦfrom CIA rats. In vitro, A771726 (0.1, 1, 10μM) suppressed the secretion function of PMΦfrom CIA rats by inhibiting LPS-TLR4 pathway.Thirdly, leflunomide could enhance the regulation of CD4+CD25+Tregs on PMΦof CIA rats. In vitro, A771726 (0.1, 1, 10μM) reinforced the suppression of TGF-βon secretion of PMΦfrom CIA rats, and enhanced the anti-inflammatory cytokine secretion. The results in RT-PCR and Western-blot indicated that A771726 strengthened the regulation of TGF-βon PMΦof CIA rats by inhibiting TLR4 expression in PMΦof CIA rats.Conclusion:①C IA rat model induced with our production of CⅡis successful as indicated the similarities in clinical manifestation, immunology and histopathology with human RA.②T he mechanisms of regulation of CD4+CD25+Tregs on PMΦof CIA rats: CD4+CD25+Tregs combined with PMΦthrough TGF-β/Tβ-RII, inhibiting TLR 4 expression of PMΦwhich leads to suppression of the immune function of LPS-stimulated PMΦof CIA rats.③Leflunomide has therapeutic effects on CIA rats, and the mechanisms contains three aspects: improving the percentage of suppressive CD4+CD25+Tregs, directly inhibiting the immune function of PMΦ, and enhancing the regulation of CD4+CD25+Tregs on PMΦby LPS-TLR4 pathway.更多还原