The stress of dry eye on corneal barrier function

描述（由申请人提供）：该项目将研究角膜上皮上紧密连接（TJ）对实验性干眼（EDE）的反应，特别强调角膜上皮屏障功能的扰动。角膜上皮屏障将被评价为俄勒冈州绿色葡聚糖（一种高分子量荧光分子（70 kda））的摄取，并将其与EDE前和后1、3、5、7、10和12天的TJ蛋白和晚期包膜蛋白（LEP）的表达相关。本研究还将评估促炎信号传导介质对响应EDE的TJ和LEP表达的作用。将评价应激相关通路/介质的特异性抑制剂在预防角膜屏障功能障碍中的有效性。通过用东莨菪碱药理学抑制泪液分泌并置于低湿度和恒定通风的环境受控室中，在小鼠中诱导干眼。将使用真实的时间PCR来评价促炎介质的基因表达。将通过激光扫描共聚焦显微镜和免疫染色来评估TJ和LEP表达。将通过蛋白质印迹、ELISA和Luminex多重测定来评价促炎介质表达。

DESCRIPTION (provided by applicant): The project will study the response of the tight junctions (TJ) to experimental dry eye (EDE) on the corneal epithelium with specific emphasis on the perturbation of corneal epithelial barrier function. Corneal epithelial barrier will be evaluated as the uptake of Oregon Green Dextran, a high molecular weight fluorescent molecule (70kda) and it will be correlated to the expression of TJ proteins and late envelope proteins (LEP) prior to and after 1,3,5,7,10 and 12 days of EDE. This study will also evaluate the role of pro-inflammatory signaling mediators on expression of TJ and LEP in response to EDE. Effectiveness of specific inhibitors of stress associated pathways/mediators will be evaluated in the prevention of corneal barrier dysfunction. The dry eye will be induced in mice by pharmacologic inhibition of tear secretion with scopolamine and placement in an environmentally controlled chamber with low humidity and a constant air draft. Real time PCR will be used to evaluate the gene expression of pro inflammatory mediators. TJ and LEP expression will be evaluated by laser scanning confocal microscopy and immunostaining. Pro inflammatory mediator expression will be evaluated by Western Blot, ELISA and the Luminex Multiplex Assay.