Biosynthesis and physiological roles of extremely long chain lipids in the eye

 描述（由申请人提供）：本项目的基本目标是研究导致睑板腺（MG）产生富脂分泌物（称为睑脂）的代谢途径。MG嵌入人和动物的睑板中。睑脂通过参与泪液膜（TF）的形成，保护眼表免受有害环境因素和干燥的影响。质量异常和/或数量不足 睑脂使人TF不稳定，这是干眼病（DE）的潜在因素之一，特别是其主要形式之一-睑板腺功能障碍。许多报告表明，DE影响美国和其他国家30%的总人口。作为我们研究的实际成果，我们希望发现新的，比现在可用的更有效的治疗DE的方法。目前，没有关于导致睑脂形成的生物合成途径的详细信息。我们的计划是用小鼠作为动物模型来填补这一重要的知识空白。早期研究发现，人和小鼠睑脂的脂质组成非常接近。此外，我们的试点实验与人类和小鼠MG组织标本表明，所有测试的脂质合成酶在两个物种中表达。因此，小鼠是用于最终靶向与MG相关的人类眼部病理学的研究的令人信服的（和方便的）模型。作为目标1，我们建议研究导致这些特定脂质形成的生物合成途径，并确定哪些酶促步骤和脂质产物对制造健康的睑脂至关重要。我们将确定在小鼠MG和附件中负责产生睑脂的主要酶的表达模式以及细胞和亚细胞定位。我们的目标2是阐明在睑，MG和附件的变化，在响应抑制或激活特定的基因相关的脂质代谢。为了测试我们的想法，我们将利用各种目前可用的基因敲除和转基因小鼠模型与已知的脂质异常，其眼部特征尚未得到表征，因为这些突变小鼠是为其他目的而开发的。此外，我们还将开发新的方法，使用反义RNA暂时沉默感兴趣的基因。作为目标3，我们将通过跟踪MG对2 H和13 C标记的膳食脂质示踪剂的摄取和代谢来确定膳食脂质是否对睑脂有重大影响。我们的目标将使用化学、生物化学、生理学和基因组方法的组合来实现，这些方法包括但不限于液相和气相色谱法、质谱法、微量热法、热台偏振光显微镜、体内共聚焦显微镜、蛋白质组学、原位杂交、免疫（组织）化学染色、基因表达分析、体内共聚焦显微镜、以及对实验小鼠眼结构的临床评价。

 DESCRIPTION (provided by applicant): The fundamental goal of this project is to investigate metabolic pathways that lead to generation of lipid-rich secretion (called meibum) by Meibomian glands (MG). MG are embedded into the tarsal plates of humans and animals. Meibum protects the ocular surface from hazardous environmental factors and desiccation through its participation in formation of the tear film (TF). Abnormal quality, and/or insufficient quantity of meibum destabilize the human TF, which is one of the underlying factors of dry eye disease (DE), particularly of one of its major forms - Meibomian gland dysfunction. Numerous reports indicated that DE affects up to 30% of the general population of the USA and other countries. As a practical outcome of our research, we expect to discover new, more effective treatments for DE than those that are available now. Currently, no detailed information on the biosynthetic pathways that lead to formation of meibum is available. Our plan is to fill in this important gap i knowledge using mice as animal models. Earlier, we have discovered that lipid compositions of meibum of humans and mice are very close. Also, our pilot experiments with human and mouse MG tissue specimens demonstrated that all tested lipid-synthesizing enzymes are expressed in both species. Thus, mice are convincing (and convenient) models for studies that are ultimately targeting human ocular pathologies related to MG. As Aim 1, we propose to investigate biosynthetic pathways that lead to formation of these specific lipids, and determine which enzymatic steps and lipid products are critical for making healthy meibum. We will determine expression patterns and cellular and subcellular localization of major enzymes responsible for producing meibum, in mouse MG and adnexa. Our Aim 2 is to elucidate changes in meibum, MG, and adnexa in response to suppression or activation of specific genes related to lipid metabolism. To test our ideas, we will make use of various currently available gene knockout and transgene mouse models with known lipid abnormalities, whose ocular features have not been characterized yet because those mutant mice were developed for other purposes. Also, we will develop new approaches for temporary silencing the genes of interest using anti-sense RNA. As Aim 3, we will determine whether dietary lipids can have a major impact on meibum by tracing the uptake and metabolism of 2H- and 13C-labeled dietary lipid tracers by MG. Our goals will be achieved using a combination of chemical, biochemical, physiological and genomic approaches, which include, but are not limited to, liquid and gas chromatography, mass spectrometry, microcalorimetry, hot stage polarized light microscopy, in vivo confocal microscopy, proteomics, in-situ hybridization, immuno(histo)chemical staining, gene expression analysis, in vivo confocal microscopy, and clinical evaluation of the ocular structures of experimental mice.