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

Toll 样受体在干眼发病机制中的作用

• 批准号: 7242931
• 负责人: RACHEL LEIGH REDFERN
• 金额: $ 10.8万
• 依托单位: UNIVERSITY OF HOUSTON
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7242931
• 关键词: 3-hydroxycholest-7-ene-14-carbaldehyde; Age; Apoptosis; Automobile Driving; Bacteria; Blindness; C57BL/6 Mouse; Cell Line; Cells; Cicatrix; Condition; Confocal Microscopy; Cornea; Cytology; Daily; Doctor of Philosophy; Dry Eye Syndromes; Epithelial Cells; Family; Flow Cytometry; Gelatinase B; Glycoproteins; Goblet Cells; Health; Human; Immunology; Infection; Inflammation; Inflammatory; Knock-out; Laboratories; Measures; Mentors; Messenger RNA; Modeling; Mus; Optometrist; Pathogenesis; Patients; Pattern; Population; Prevention; Principal Investigator; Production; Quality of life; Reaction; Reading; Reporting; Research; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Risk; Role; Sampling; Simian virus 40; Sjogren' s Syndrome; Source; Stimulus; Stress; Symptoms; TLR2 gene; Testing; Therapeutic Intervention; Time; Toll-Like Receptor 2; Toll-like receptors; Training; Training Programs; Tumor Necrosis Factor-alpha; Ulcer; Virus; Work; chemokine; conjunctiva; corneal epithelium; cytokine; experience; eye dryness; fungus; human TNF protein; human subject; impression; insight; irritation; multidisciplinary; novel; ocular surface; programs; protein expression; receptor; receptor expression; response; skills

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.

描述（由申请人提供）：拟定研究计划的主要目的是为主要研究者（PI）提供发展成为眼表炎症独立研究者所需的全面教学和实验室培训。多学科培训计划将在导师Alison McDermott博士和Stephen Pflugfelder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，该拟定研究项目将通过研究扩大其先前的科学经验，以研究Toll样受体（TLR）在干眼综合征眼表炎症中的作用。TLR的活化已显示刺激细胞因子和趋化因子的产生， 引发炎症反应该提案的科学重点将调查TLR刺激干眼综合征炎症的一般假设。具体目标是：（1）建立TLR 2、4、5和9在来自轻度、中度和重度干眼症、干燥综合征和年龄匹配的正常受试者的人结膜上皮细胞中的表达模式。将从人类受试者中采集结膜印迹细胞学样本，并分别通过真实的时间RT-PCR和流式细胞术测定TLR mRNA和蛋白表达;（2）确定TLR 2，4，5和9在原代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对高渗应激和细胞因子的应答通过真实的时间RT-PCR和共聚焦显微镜观察治疗前后小鼠的TLR 2、4、5和9的表达情况;（3）比较对照组和干燥刺激诱导的实验性干眼症（EDE）小鼠TLR 2、4、5和9的表达情况。此外，该特定目的将确定EDE TLR 2、4和9缺陷型小鼠是否具有减少的眼表面炎症，如通过a.）MMP-9和细胞因子（IL-1 β和TNF-α）mRNA和蛋白质表达的降低和B.）与野生型EDE C57 BL/6小鼠相比，上皮细胞凋亡和杯状细胞损失。 这些研究将有助于了解TLR在干眼发病机制中的作用，同时也为PI提供培训计划，以培养成为成功的独立研究者所需的技能。