树鼩复发慢性化实验性自身免疫性葡萄膜炎（EAU）模型研究

Intraocular chronic-relapsing inflammation in human autoimmune uveitis is a main factor contributed to blindness in patients with uveitis, but so far the underlining mechanisms of disease recurrences have not been fully elucidated. A major limitation of experimental autoimmune uveitis (EAU) in mice is that they do not minic the chronic-relapsing course in human autoimmune recurrent uveitis. The immune responses of EAU in rats were not identical to those in human autoimmune recurrent uveitis. We have developed a chronic-relapsing EAU in B10RIII mice induced by interphotoreceptor retinoid-binding protein 161-180 in our recent study, but the relapsing inflammation were observed only once. We therefore set out to development a model of chronic-relapsing EAU in Chinese tree shrew, which is regarded as close relative of non-human primates and is similar to human in visual system and immune system. The project mainly includes 4 parts as follow. (1) The chronic-relapsing EAU in Chinese tree shrew will be induced by immunization with retinal soluble antigen(S-Ag), IRBP in CFA as well as by the transfer of activated, antigen-specific T cells to determine the influence of variables, such as the type and dose of antigen and methods of immunization on chronic-relapsing disease. (2) The clinical findings, incidence and severity and histopathology of relapsing EAU will be studied to identify the characteristics of chronic-relapsing disease obtained under the different conditions of immunization. (3) The freqencies and functional of Th1 and Th17 and Treg will be assessed to study the immune reponse characteristics of relapsing EAU. (4) Disturbtion of chronic-relapsing EAU will be investigated to test the model for its practicality and effectiveness in drug evaluation and development. These studies are expected to gain a deep insight into the initiating mechanisms involved in uveitis and to provide a new strategy in the prevention and treatment for this disease.

炎症的复发和慢性化是致葡萄膜炎患者盲目的根本原因。现有小鼠实验性自身免疫性葡萄膜炎（EAU）模型难以模拟这一本质特征，大鼠复发性EAU免疫机制与人类葡萄膜炎也不尽相同。最近我们预实验建立了小鼠复发慢性化EAU，但仅出现1次复发。本项目以在视觉和免疫系统与人类高度相似的灵长类近亲树鼩为研究对象，探讨不同视网膜抗原（S-Ag，IRBP）及其致敏的T细胞诱导葡萄膜炎发生、复发及慢性化的条件，筛选诱导树鼩复发慢性化EAU的特异免疫类型、剂量和方式；评价上述筛选出树鼩复发性EAU在疾病发生、高峰、消退和复发时的临床表现、炎症严重程度和病理组织学，鉴定复发模型的临床表现和病理特征；通过评价模型中效应性Th1和Th17及调节性T（Treg）在疾病发生、消退和复发的功能和数量，探讨复发性EAU免疫反应特征；评价干预树鼩复发性EAU来探讨模型应用及示范。旨在为葡萄膜炎发生及防治提供新的理想策略。

炎症的复发和慢性化是致葡萄膜炎患者盲目的根本原因。现有小鼠EAU模型难以模拟这一本质特征，大鼠复发性EAU免疫机制与人类葡萄膜炎也不尽相同。本项目以灵长类近亲树鼩为研究对象，利用预测的树鼩IRBP182-192、R14和IRBP1197-1211以及人和牛R14、R16等视网膜抗原免疫树鼩，筛选树鼩复发慢性化EAU发生条件和特征。结果发现，人、牛R14以及树鼩IRBP182-192、R14和IRBP1197-1211可成功诱导树鼩EAU，其中50-800µg牛R14和IRBP1197-1211可诱导树鼩复发慢性化全葡萄膜炎，免疫后6-15天发病，25天左右达炎症高峰，后炎症慢性化，至观察期253天仍可见炎症；临床表现结膜或睫状发生-消退反复性充血，角膜损伤，瞳孔变形，虹膜粘连。眼底见视网膜变性，血管迂曲；组织病理学特征为慢性全葡萄膜炎：炎症累及结膜、角膜、葡萄膜和视网膜，角膜损伤，虹膜脉络膜血管充盈，包括色素上皮在内的视网膜结构紊乱、折叠弯曲，色素上皮与光感受器层脱离，嗜酸性物沉积；免疫表型表现表达CD45、CD68和Iba-1的白细胞、巨噬细胞广泛浸润于结膜、角膜、葡萄膜和视网膜，光感受器层、内核层和节细胞层散在浸润CD4+T细胞和极个别CD8+T细胞；差异基因组研究发现，牛R14诱导的EAU中共表达991个差异转录本，其基因本体论GO归类显著富集于鸟苷酸（cGMP）环化酶活性；IRBP1197-1211诱导EAU中共表达1046个差异转录本，其中参与光感受器正常生理功能的G蛋白信号调控子4（RGS4）显著性上调。RGS4抑制剂CCG203769和双氢青蒿素能通过促进CD8+T细胞浸润降低EAU病理损伤。通过上述研究，发表被眼科或免疫学同行专家认同的SCI文章3篇，CSCD文章1篇，另外1篇SCI文章待发表，可为葡萄膜炎发生及防治研究提供新的疾病模型。参加学术会议8人次，培养硕士研究生6名，学术青年3名。