Proteins in the Molecular Mechanisms of Tear Film Formation

项目总结/摘要 这一建议的前提是，目前a3的教条缺乏生物化学证据 分层泪膜模型。干眼病的治疗需要了解其分布和相互作用 组成泪膜结构的生物成分。目前，缺乏治疗的科学依据。 替换缺失的组件或支持以某种方式起作用的组件的策略， 一直是干眼症成分的基础，但效果并不好。这种方法是有缺陷的，直到地点和 确定组件的功能。在泪膜正常之前， 结构理解。脂质和蛋白质泪液的分布已从非特异性 没有生化证据的干涉测量数据。了解这些生物分布的一个主要障碍 然而，缺乏跟踪单个分子及其功能相互作用的技术一直是这些分子的主要组成部分。的 泪膜的脂质层厚度为数十纳米。解决方案中动态组件的直接可视化 至今仍是科学家们无法企及的。该项目将利用最近的技术进步， 荧光显微镜和荧光光谱法来跟踪脂质和蛋白质。两个提案目标是 旨在显示泪膜中脂质和蛋白质的主要成分的分布。第一个目标 将使用定制的亚衍射（“超分辨率”）显微镜来定位隔间中的荧光团 的眼泪在9 nm的分辨率。该显微镜是专门为泪膜结构而构建的。第二 aim建议采用高灵敏度的单光子计数荧光测量来解决 单个分子在亚纳米距离上的相互作用。我们的实验室已经完善了设备， 设备、试剂和技术专长来进行这些测量。这项提案的成功将导致 组成泪液的主要脂质和蛋白质分布的生化数据。这项研究可能会取代 具有科学基础的模型，用于测试泪膜成分的功能并创建干眼症解决方案 疾病

Project Summary/Abstract The premise of this proposal is that there is a dearth of biochemical evidence for the current dogma of a 3 layered tear film model. The treatment of dry eye disease requires knowledge of the distribution and interactions of biologic components that comprise tear film structure. Currently, the scientific basis for treatment is lacking. The strategy of replacement of absent component or bolstering components that function in a certain way has been the basis for ingredients for dry eye and it has not worked well. This approach is flawed until locations and functions of components are determined. The need for better treatment can not be met until normal tear film structure is understood. The distribution of lipids and proteins tears has been presumed from non-specific interferometry data without biochemical proof. A major obstacle to understanding the distribution of the components has been the lack of technology to track individual molecules and their functional interactions. The lipid layer of tear film is tens of nanometers in thickness. Direct visualization of dynamic components in solution was until now beyond the reach of scientists. This project will capitalize on recent technological advances in fluorescence microscopy and fluorescence spectroscopy to track lipids and proteins. Two proposal aims are designed to show the distribution of the major components of lipids and proteins in the tear film. The first aim will use customized sub-diffraction ("super-resolution")microscope to localize fluorophores in the compartments of tears at a resolution of 9 nm. The microscope was specifically constructed for tear film structure. The second aim proposes to incorporate highly sensitive single photon counting fluorescence measurements to resolve interactions of individual molecules at sub-nanometer distances. Our laboratory has honed the facilities, equipment, reagents and technical expertise to make these measurements. Success with this proposal will lead to biochemical data for the distribution of major lipids and proteins that comprise tears. The research may replace models with a scientific foundation to test functions of the tear film components and create solutions for dry eye diseases.