Graves病中IL-10基因变异及其影响CD4+CD25+调节性T细胞诱导分化的功能及机制初探

Graves' disease (GD) is the most common organ-specific autoimmune disorder with multiple genetic predispositions characterized by reactivity to self-thyroid antigens and lymphocytic infiltrate within the thyroid. The hereditary susceptibility and immune complex deposition are believed to contribute to this disease, but its etiology remains unclear. There is strong evidence that the members of the interleukin family have participated in mediating inflammation, which may affect the outcome of patients with GD. We previously found that the single nucleotide polymorphisms (SNP) of many Interleukin genes were associated with GD and Graves' ophthalmopathy (GO) susceptibility in Chinese population. Especially, the IL-10 gene was the most significant one, and the serum level of IL-10 were increased in GD and GO patients, which suggested that IL-10 gene may contribute to GD development (In the work basis). A large body of recent studies have highlighted the importance of pathogenesis induced CD4+CD25+Treg cells and IL-10 in immuno-suppression in Graves' disease. So on this study, we firstly investigate the definite role of gene polymorphism and anti-inflammatory activity of IL-10 in Graves' disease. According to the genetic analysis of wild type and mutant reporter plasmids of the IL-10 gene transcription activity, we know the phenotype-genotype correlation of IL-10 with molecular genetic of Graves' disease. Secondly we study the quantity of CD4+CD25+Treg cells in peripheral blood of Graves'disease and explore the mechanisms of the potential regulatory role of IL-10 on CD4+CD25+Treg cells. So, we enrolled about 200 cases of Various stages of GD patients and healthy donors as normal controls, detected The proportions of CD4+CD25+Treg cells by flow cytometric analysis and immunosuppression activity by 3H-TdR.Then by investigating the effect of Transforming growth factor-β (TGF-β), associated signaling on the expression of forkhead box protein-3 (Foxp3), and a selective IL-10 siRNA, we know whether IL-10 signals is needed to maintain expression of the Foxp3 and suppressive function of Treg cells. Finally, we establish an in vitro experimental model of Thyroid follicular cells (TFCs) and CD4+CD25+T lymphocyte cells by co-culture technique. In this model, T-cell mediated inflammatory responses of thyroid follicular cells were regulated by using the skills of Real-time PCR and ELISA to detect the cytokines such as IL-2、6、10, IFN-γ and TGF-β secretion. Thyroid hormones production and thyroglobulins (Tg) release were analyzed by radioimmunoassay. It is of important value to study the role of IL-10 and Treg cell signals in immune regulation, for the illustration of immunological pathogenesis and exploration of new means to immune intervene in Graves' disease.

Graves病（GD）是一种多基因遗传自身免疫性疾病，病因尚未明了，当前研究热点侧重基因遗传及免疫紊乱的致病机制。我们前期工作发现IL-10基因启动子多态性位点与GD及Graves眼病（GO）发病风险相关，并且IL-10在GD及GO中表达升高，提示IL-10可能是GD发生的重要易感基因（见工作基础）。近年来研究表明在GD的发生发展中IL-10及CD4+CD25+调节性T细胞（Treg）是重要的免疫调节因子，本研究在进一步确定IL-10基因多态性及其作为抗炎因子在GD中的作用基础上，探讨IL-10调控Treg细胞炎症反应的分子机理，及其对重要转录因子Foxp3的信号调节通路的影响，并建立具有自身免疫特性的GD甲状腺滤泡细胞和T淋巴细胞体外复合培养细胞模型，探讨IL-10及Treg细胞调控甲状腺滤泡细胞自分泌及旁分泌相关细胞因子介导免疫耐受的机制，为GD有效防治提供新思路和药物作用的新靶点。

Graves病是一种多基因遗传自身免疫疾病，本实验组前期工作通过分析病例-对照人群中白介素系列基因snp多态性位点，发现IL-10基因启动子区域rs1800896(G-1082A)多态性与Graves病及Graves眼病易感性具有显著的相关性，我们进一步实验发现，该位点G等位基因增加Graves病的发病风险，而A等位基因可能具有保护作用。收集未治初发Graves病、合并突眼、难治性、复发性、临床缓解型Graves病、正常对照组，各30-50例，检测各组CD4+CD25+Treg细胞、血清IL-10水平，发现IL-10与Treg细胞的成反相关性，疾病各组Treg细胞均低于正常对照组，IL-10相反，疾病组血清中IL-10含量较对照组升高，并且随着疾病的不断好转，Treg细胞含量逐渐升高，而IL-10的含量逐渐下降，通过硒元素可调节机体内IL-10及Treg细胞的表达，硒元素可减少机体炎症因子、IL-10的含量，使Treg细胞增加，初步鉴定可能是通过调节转录因子c-Rel活性相关的信号通路而实现的。IL-19、IL-20、IL-22、IL-26是IL-10家族成员，我们前期通过上海人群病例对照研究已发现该四种白介素基因多态性位点在两组人群中具有显著性差异，本课题组再次收集大连地区Graves病及正常对照人群各400余例，再次验证该四种白介素基因snp位点两组间具有明显的统计学差异，血清学同时显示该四种白介素因子与TRAb水平具有同源性，与疾病所处的不同程度有密切相关，以IL-26最甚。提示IL-10及IL-10家族与Graves病发病及疾病的转归有密切的相关性，IL-26有望成为Graves疾病诊断及治疗评判的新的炎症指标。