The Role of Toll-Like Receptors in the Pathogenesis of Dry Eye

描述(由申请人提供)：拟议研究计划的主要目标是为首席调查员(PI)提供必要的全面教学和实验室培训，以发展为眼表炎症的独立研究人员。多学科培训计划将在导师艾莉森·麦克德莫特博士和斯蒂芬·普弗洛格费尔德医学博士的指导下进行。PI是一名具有角膜免疫学背景的验光师，这项拟议的研究计划将通过研究Toll样受体(TLRs)在干眼综合征眼表炎症中的作用来扩展她以前的科学经验。TLRs的激活已被证明能刺激细胞因子和趋化因子的产生， 引发炎症反应。这项提案的科学重点将调查TLRs刺激干眼综合征炎症的一般假设。目的：(1)建立轻度、中度、重度干眼症患者、干燥综合征患者及年龄匹配的正常人结膜上皮细胞TLR2、4、5、9的表达模式。方法：(1)收集受试者结膜印迹细胞学标本，分别采用实时荧光定量RT-PCR和流式细胞术检测TLR2、4、5和9在原代培养的人结膜和角膜上皮细胞以及正常人结膜和角膜上皮细胞系中的表达及定位模式；(3)比较TLR2、4、5和9在正常小鼠结膜和角膜上皮细胞系中的表达规律；(3)比较对照组C57BL/6小鼠与干燥刺激C57BL/6诱导的实验性干眼眼(EDE)小鼠TLR2、4、5和9的表达模式。此外，这一特定目标将确定EDE TLR 2、4和9缺陷小鼠是否减少了眼表面炎症，通过a.减少基质金属蛋白酶-9和细胞因子(IL-1β和肿瘤坏死因子-α)的mRNA和蛋白表达，以及b。与野生型EDE C57BL/6小鼠比较，上皮细胞凋亡和杯状细胞丢失。这些研究将有助于了解TLRs在干眼发病机制中的作用，同时也为PI提供培训计划，以发展成为一名成功的独立研究人员所需的技能。

DESCRIPTION (provided by applicant): The primary objective of the proposed research plan is to provide the Principal Investigator (PI) with the comprehensive didactic and laboratory training necessary to develop into an independent researcher in ocular surface inflammation. The multidisciplinary training plan will be under the guidance of mentors Alison McDermott, PhD and Stephen Pflugfelder, MD. The PI is an Optometrist with a background in corneal immunology and this proposed research program will expand upon the her previous scientific experience through studies to investigate the role of Toll-like receptors (TLRs) in ocular surface inflammation in dry eye syndrome. The activation of TLRs has been shown to stimulate the production of cytokines and chemokines, initiating an inflammatory reaction. The scientific focus of this proposal will investigate the general hypothesis that TLRs stimulate inflammation in dry eye syndrome. The specific aims are: (1) To establish TLR 2, 4, 5 and 9 expression pattern in human conjunctival epithelial cells from subjects with mild, moderate, and severe dry eye, Sjogren's syndrome, and age matched normal subjects. Conjunctival impression cytology samples will be collected from human subjects and TLR mRNA and protein expression will be determined by real time RT-PCR and flow cytometry respectively; (2) To determine the expression and localization pattern of TLR 2, 4, 5 and 9 in primary human conjunctival and corneal epithelial cells and in normal human conjunctival and corneal epithelial cell lines in response to hyperosmolar stress and cytokine treatment by real time RT-PCR and confocal microscopy; (3) To compare the TLR 2, 4, 5 and 9 expression pattern among control C57BL/6 mice with experimental dry eye (EDE) induced by desiccating stimulus C57BL/6 mice. Additionally this specific aim will determine if EDE TLR 2, 4, and 9 deficient mice have reduced ocular surface inflammation as measured by a.) a reduction in MMP-9 and cytokine (IL-1beta and TNF-alpha) mRNA and protein expression and b.) epithelial cell apoptosis and goblet cell loss compared to wild type EDE C57BL/6 mice. These studies will help understand the role of TLRs in the pathogenesis of dry eye while also providing the PI with a training program to develop the skills necessary to become a successful independent investigator.