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

极长链脂质在眼中的生物合成和生理作用

基本信息

批准号：
9106468
负责人：
Igor A Butovich
金额：
$ 40.49万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-08-01 至 2020-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9106468
关键词：
Affect Anabolism Animal Model Animals Ankle Antisense RNA Attention Biochemical Biochemical Pathway Blood Cells Characteristics Chemicals Chemistry Cholesterol Confocal Microscopy Consensus Country Data Desiccation Detection Diet Dietary Fats Disease Environmental Risk Factor Enzymatic Biochemistry Enzymes Equilibrium Evaluation Eye Eye diseases Eyelid structure Fatty Acids Fatty Alcohols Film Gene Expression Profile Gene Expression Profiling Gene Mutation Gene Silencing General Population Generations Genes Genetic Genomic approach Goals Health Human In Situ In Situ Hybridization Individual Knockout Mice Knowledge Label Laboratories Lead Length Lipids Maps Mass Fragmentography Metabolic Pathway Metabolism Methods Microarray Analysis Morphology Mus Mutant Strains Mice Mutation Nature Ocular Pathology Oleic Acids Outcome Pathology Pathway interactions Pattern Physiological Physiology Polarization Microscopy Population Procedures Production Proteins Proteomics RNA Reporting Research Role Sampling Small Interfering RNA Specimen Stable Isotope Labeling Staging Staining method Stains Structure Study models Symptoms Tarsal plate Testing Therapeutic Intervention Tissues Tracer Transgenes Validation Wild Type Mouse Work effective therapy eye dryness fatty acid biosynthesis human disease human subject hydroxy fatty acid in vivo interest knockout gene lipid biosynthesis lipid metabolism meibomian gland meibomian gland dysfunction microcalorimetry mouse model novel strategies novel therapeutics ocular surface pi bond public health relevance research clinical testing research study response targeted treatment technique development treatment strategy uptake

项目摘要

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.

描述（由申请人提供）：该项目的基本目标是研究导致嵌入人类和动物睑板腺（MG）产生富含脂质的分泌物（称为睑脂）的代谢途径。睑脂通过参与泪膜 (TF) 的形成，保护眼表免受有害环境因素和干燥的影响。睑板腺破坏人类 TF 的稳定性，这是干眼病 (DE) 的根本因素之一，尤其是其主要形式之一——睑板腺功能障碍。大量报告表明，高达 30% 的美国普通人群患有干眼症。作为我们研究的实际成果，我们期望发现比现有方法更有效的新治疗方法，目前还没有关于导致睑脂形成的生物合成途径的详细信息。我们的计划是使用小鼠作为动物模型来填补这一重要的空白，此前，我们发现人类和小鼠的睑脂成分非常接近，而且我们对人类和小鼠 MG 组织样本的初步实验表明，所有这些都与人类和小鼠的睑脂成分非常接近。因此，小鼠是最终针对与 MG 相关的人类眼部病理学研究的令人信服的（且方便的）模型，我们建议研究导致形成的生物合成途径。我们的目标 2 是阐明小鼠 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。为了测试我们的想法，我们将利用各种目前可用的具有已知脂质的基因敲除和转基因小鼠模型。其眼部特征尚未确定，因为这些突变小鼠是为其他目的而开发的。此外，我们将开发使用反义 RNA 暂时沉默感兴趣基因的新方法，作为目标 3，我们将确定饮食中的脂质是否存在。通过 MG 追踪 2H 和 13C 标记的膳食脂质示踪剂的摄取和代谢，可以对睑脂产生重大影响。我们的目标将通过化学、生化、生理和基因组方法的组合来实现，其中包括，但不限于液相和气相色谱法、质谱法、微量热法、热台偏光显微镜、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，以及实验小鼠眼部结构的临床评价。