警报素HMGB1介导的固有免疫参与真菌性角膜炎调控机制的研究

Fungal infection of the eye are important diseases, even with keratoplasty, the prognosis is poor. And the incidence is raising nowaday in China, while the mechanism is not clear. Alarmin HMGB1 is one of the key proinflammatory factors in innate antifungal immunity, which was released from inflammatory cells, followed by combining with receptors, then regulated the innate and acquired immunity. Recently, we found that the expression of HMGB1 in keratitis tissue was upregulated, and in the culture media, the concentration was higher than that from the cultured human limbal epithelial cells when challenged by Candida albicans. This study systematically investigates the mechanism of HMGB1 involving in fungal keratitis, by corneal epithelial cells and fungi co-culture system and animal model, to block or interfere with HMGB1 from different levels, in order to analyze the mechanism of HMGB1 related molecules in fungal keratitis. And from macro and micro angles of view, we tend to understand its effect on the course of the disease and the mechanism for the pathogenesis of fungal keratitis. At the same time, our aim is by inhibiting the activity of HMGB1 and reducing the production of related inflammatory factors, to reduce the inflammatory reaction of corneal tissue. If achieved, the role of HMGB1 in host anti-fungal defence and inflammatory response during fungal keratitis will be well established. And this study will afford a novel therapeutical benefit in the treatment of fungal keratitis.

真菌性角膜炎是较常见的难治性致盲眼病，发病率在我国有上升趋势，即使联合角膜移植手术，预后仍较差。警报素HMGB1介导固有免疫的作用日益引起重视，其在细胞外发挥警报素作用，与免疫细胞表面特定受体结合，对固有免疫和获得性免疫进行调节。本组前期实验发现HMGB1在真菌性角膜炎组织表达增强，角膜上皮细胞受到真菌刺激后，其胞浆转移，且培养液中含量增加。本研究采用真菌性角膜炎动物模型和角膜上皮细胞与致病真菌共培养体系，系统研究HMGB1及相关分子参与真菌性角膜炎发病机理，通过不同层次分别阻断或干扰HMGB1，判定其参与该疾病的相关机制，从宏观和微观角度了解HMGB1对病程的作用，为真菌性角膜炎发病机制提供实验依据。在此基础上通过抑制HMGB1的活性，减少其相关炎症因子的产生，降低真菌性角膜炎的炎症反应，为其生物治疗提供一种新思路。

感染性角膜病是重要的全球性致盲眼病之一，在严重的角膜溃疡病例中，真菌性角膜炎占很大比例，特别是在发展中国家。患者多集中在户外人群，往往是青壮年。深入了解真菌性角膜炎的发生机制，开发新型治疗方法是当务之急，寻求真菌性角膜炎新型生物治疗靶点是研究热点之一。.HMGB1是目前多种疾病研究的热点和治疗的靶点之一。其不仅是免疫学研究的热点之一，而且逐渐成为多种疾病治疗的新型生物靶点，尤其是炎症与感染性疾病，在真菌性角膜炎发病过程中，HMGB1很可能对疾病的发生及进展产生调节效应，从更深的层次和更高的方向揭示真菌性角膜炎的发病机制，使其成为眼科炎症治疗的新型生物靶点。本组率先将其应用于真菌性角膜炎发病机制研究中，通过分子生物学及组织免疫学研究，证实警报素HMGB1参与真菌性角膜炎发病过程中的固有免疫反应，从体内及体外实验发现受到致病真菌侵袭后，HMGB1具有特定的变化及发展规律；在真菌性角膜炎发病过程中，HMGB1的变化与真菌性角膜炎炎症严重程度及疾病的发展转归存在着密切的相关性；综合本课题各部分实验结果，系统阐明HMGB1参与真菌性角膜炎的发病机理，为真菌性角膜炎治疗提供新的思路。.采用小鼠实验性角膜炎模型和人角膜上皮细胞（HCECs）体外培养模型，探讨活性氧（ROS）诱导的氧化应激在真菌性角膜炎发病机制中的作用。真菌角膜和接触白色念珠菌的HCECs中ROS的产生明显增加，并伴有p38丝裂原活化蛋白激酶（MAPK）的激活。研究结果首次证明ROS诱导的氧化损伤通过p38mapk途径参与真菌性角膜炎的发病机制，为真菌性角膜炎的潜在治疗提供了新的靶点。病理条件下，中、高浓度的活性氧（ROS）通过细胞氧化应激反应可以诱导细胞凋亡甚至导致其坏死。ROS与细胞信号转导之间也存在内在关联。AIP1/NOX4/NLRP3、NLRP6炎症小体可以直接调控角膜炎症和角膜新生血管。