糖尿病视网膜病变（DR）是严重损害视功能的最常见眼科疾病之一。白细胞粘附和炎性损伤是DR特征性表现，但具体发生机制尚不清楚。中性粒细胞胞外陷阱（NETs）是一种新发现的炎症作用形式，前期研究发现NETs与DR关系密切，为DR研究提供了新方向。本研究拟比较DR患者和正常人血及眼内的NETs表达差异；通过中性粒细胞和视网膜微血管内皮细胞共培养和屏障建立，明确NETs在中性粒细胞粘附中的角色，阐明NETs对微血管内皮细胞和微血管屏障的损伤作用及主要效应成分；建立小鼠DR模型,观察视网膜血管中NETs粘附情况及对血管功能的影响；通过对肽酰精氨酸脱亚氨酶-4（PAD4）表达的体内和体外干预，阐明PAD4调控NETs形成参与DR炎性损伤的机制及对DR病程的影响。本项目将明确NETs在DR炎性损伤中的作用，寻找NETs调控因子为开发有效防治DR的新药提供理论依据，具有重要意义和科学价值。

Diabetic retinopathy (DR) is one of the most severe eye diseases which would irreversibly destroy visual function. Up to now, leukocyte adhesion and inflammation have been considered as the key elements for the pathogenesis of DR. However, the mechanism of how leukocytes adhere to endothelial cell and induce inflammation is still not clear. Recently, a novel mechanism for neutrophil inducing inflammation was described as neutrophil extracellular traps (NETs).Interestingly, our research found that NETs was related to DR, which provide a new direction for preventing and treating diabetic retinopathy. In the present project, we plan to do researches to explore the effect of NETs on leukocyte adhesion and inflammatory microvascular injury, also how it is regulated. Firstly, we plan to compare the existence of NETs in blood and eye between patients with and without diabetic retinopathy. Secondly, we plan to explore the effect of high glucose and advanced glycation end products（AGEs) on NETs formation ,and whether peptidyl arginase deaminase 4 (PAD4) are involved. Thirdly, we plan to build mice retinal microvascular endothelial cells (RMECs) and neutrophil co-culture model, to explore whether neutrophil adhere to RMECs mainly as NETs, to analyze the effect of NETs on RMECs viability and apoptosis by inhibiting NETs formation with PAD4 inhibitor or digesting NETs with DNase. We would also build RMECs barrier model, to explore the effect of NETs on RMECs barrier integrity and the expression of tight junction proteins, and to figure out whether this effect is elastase, histone and myeloperoxidase dependent by using antagonists. At the meantime, we plan to build diabetic retinopathy model in PAD4 wild type and PAD4 knock out mice, to explore the effect of PAD4 on NETs mediated neutrophil adhesion and inflammatory microvascular injury. This project would explore the role of NETs in microvascular barrier integrity and the pathogenesis of DR, would provide novel theoretical basis for developing better method to prevent and treat diabetic retinopathy, which is with scientific value and clinical significance.