miR-155和miR-146a对甲状腺相关眼病的免疫及增殖反应的调节作用

The immune and proliferative response, caused by the CD4 T cells and orbital fibroblasts (OF) are important factors in thyroid associated ophthalmopathy (TAO). miR-155 can induce the activation of T cells and boost the immune response; while miR-146a inhibits T cell activation and negative regulate the immune response; and both were involved in cell proliferation and differentiation process. Our previous work found: the expression of miR-146a was decreased in the orbital tissue and PBMC in TAO patients, while the expression of miR-155 was increased. But the expression，function and mechanism of miR-146a and miR-155 in CD4 T cells and OF in TAO patients were still not clear. Using PCR, RNA transfection, Flow cytometry, Western blot, ELISA, mixed culture,immunoprecipitation and gene sequencing, we will try to clarify: the aberrant expression of miR-146a and miR-155 in CD4 T cells and OF; and their role in the regulation of immune and proliferative response in thyroid associated ophthalmopathy; their target genes and the molecular mechanisms of the abnormal expression. From the new viewpoint of microRNA, this study is to explore the pathogenesis of TAO, and lay the foundation for the prevention and treatment of TAO.

CD4+T细胞、眼眶成纤维细胞（OF）参与的免疫及增殖反应，是甲状腺相关眼病（TAO）发生的重要因素。研究表明：miR-155诱导活化T细胞，促进免疫反应；而miR-146a抑制T细胞的活化，负向调控免疫反应；且两者均参与细胞增殖分化等过程。我们前期发现：miR-146a 在TAO 眶组织及PBMC中低表达，而miR-155高表达。但miR-146a和miR-155在TAO患者CD4+T细胞、OF中的水平，以及它们的作用及机制尚不明确。我们拟采用PCR、RAN转染、流式细胞、Western blot、ELISA、混合培养、免疫沉淀、基因测序等手段，从分子及细胞水平阐明miR-155和miR-146a在CD4+T细胞、OF中的异常表达及其对TAO免疫及增殖反应的影响；确定其靶基因及其异常表达的分子机制。本研究将从microRNA这个新视点探索TAO的发生机制，为TAO的防治奠定基础。

甲状腺相关性眼病（thyroid-associated ophthalmopathy,TAO)，是一种器官特异性自身免疫性疾病，多与甲状腺疾病有关，其确切病因和发生机制尚不清楚。目前研究表明，CD4+T细胞和眼眶成纤维细胞（orbital fbroblasts，OF）参与的免疫和及增殖反应在TAO的发生发展中起重要作用。microRNA通过降解靶基因或者抑制转录后的翻译水平，进而影响细胞的分化、增殖和凋亡，并在生物生长发育和疾病发生发展过程中发挥重要的调节作用。本课题围绕miR-155和miR-146a对甲状腺相关眼病的免疫及增殖反应的调节作用展开系列实验研究，我们发现：miR-146a和miR-155在甲状腺相关眼病眼眶成纤维细胞中较正常对照组呈低表达，过表达miR-146a可抑制眼眶成纤维细胞中IL-6和IL-8的表达，进而抑制眼眶成纤维细胞的增殖反应。miRNA-146a和miRNA-155在甲状腺相关眼病CD4+T中细胞较正常对照组呈低表达。miR-146a或miRNA-155抑制表达可以提高CD4+T细胞增殖活性；上调共刺激分子CD40L的表达；促进细胞因子IL-10,TGF-β的表达；上调B细胞产生 IgG的能力，促进NF-KB信号通路中IRAK1和TRAF6的表达，因此我们认为：miRNA-146a和miRNA-155在甲状腺相关眼病CD4+T中低表达，可上调CD4+T细胞增殖活性及相关免疫分子的表达，并通过NF-KB信号通路促进甲状腺相关眼病的免疫反应。进一步通过荧光素酶报告基因系统，检测miR-155和miR-146a在甲状腺相关眼病CD4+T细胞中的靶基因。发现NUMB为miRNA-146a的靶基因；SPI1是miRNA-155的靶基因。通过以上研究，我们基本明确了miR-155和miR-146a在TAO患者的CD4+T细胞和OF的异常表达，及其对免疫和增殖的调节作用，为阐明TAO的发病机制和防治提供基础。