Lipogenesis in the meibomian glands and adnexa in the norm and pathology

正常和病理情况下睑板腺和附件的脂肪生成

• 批准号: 9762911
• 负责人: Igor A Butovich
• 金额: $ 40.5万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-30 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9762911
• 关键词: Adhesions; Affect; Aftercare; Age; Aging; Anabolism; Antibodies; Biochemical; Biochemistry; Cell surface; Characteristics; Chemicals; Cholesterol; Complex; Development; Disease; Dose; Dyslipidemias; Enzymes; Equilibrium; Eye diseases; Fatty Acids; Film; Functional disorder; Gender; Gene Expression; Gene Expression Profile; Generations; Genomics; Global Change; Goals; Grant; Health; High Pressure Liquid Chromatography; Human; Immunohistochemistry; Impairment; In Situ; In Situ Hybridization; Incidence; Intake; Intervention; Lead; Link; Lipids; Mammals; Maps; Mass Spectrum Analysis; Medical; Menopausal Status; Metabolic Diseases; Microscopy; Molecular; Morphology; Mus; Nutritional Study; Ocular Pathology; Ocular Physiology; Pathology; Pathway interactions; Patients; Pharmacologic Substance; Phosphatidylcholine-Sterol O-Acyltransferase; Physiology; Polarization Microscopy; Polysaccharides; Property; Protein Analysis; Proteins; Proteomics; Quality of life; RNA analysis; Regulation; Reporting; Research; Role; Rupture; Sampling; Scheme; Severities; Stains; Sterol O-Acyltransferase; Structure; Testing; Tissues; Vision; Western Blotting; Woman; Work; amphiphilicity; base; biophysical properties; biophysical techniques; blood lipid; clinical Diagnosis; experimental study; eye dryness; immunocytochemistry; innovation; lipid biosynthesis; lipid metabolism; meibomian gland; meibomian gland dysfunction; melting; metabolic abnormality assessment; metabolomics; microcalorimetry; ocular surface; public health relevance

Research Abstract The goal of this project is to elucidate the enzymatic pathways that underlie the biosynthesis of lipids that are produced by human meibomian glands (HMG) and are collectively known as meibum. Meibum is a critically important part of the tear film (TF) that, among other functions, protects the ocular surface from desiccating. Meibum is formed predominantly from a unique mixture of very, and extremely, long chain complex lipids. Our previous projects were devoted to biochemical and biophysical characterization of meibomian lipids (ML). Now we propose to investigate the biosynthetic pathways that lead to the formation of meibum in HMG. The following three lines of research are proposed: (I). Despite recent progress in the characterization of the lipidomes of meibum and TF, there is no clear understanding of the biosynthetic path- ways involved in the biosynthesis of meibum. The unusual and, often, unique qualities of ML imply their in situ and/or de novo origination, which, in turn, requires proper molecular mechanisms to be implemented in HMG. We will study the levels of expression, and map, major lipid-metabolizing enzymes and related proteins, which, based on our preliminary studies, are expected to be present in HMG. This will be achieved by using a combination of approaches, such as microarray RNA analyses, immunohistochemistry/immunocytochemistry, in-situ hybridization, proteomics, and lipidomics. (II). Recent reports indicate that high total blood lipid levels, including cholesterol and TAG, are correlated with higher incidence and/or severity of MG dysfunction. Also, a positive impact of ω3 fatty acids (ω3FA) supplements on the ocular surface was reported for humans and mice. Yet, the reasons and molecular mechanisms of these effects are unknown. We will determine the balance between the in situ/de novo biosynthesis of ML and the levels and composition of blood lipids. We will test if 1) enhanced intake of pharmaceutical doses of ω3FA, and (2) use of statins, can have a positive impact on the biosynthesis, composition and properties of meibum. (III). Recently, we reported that: (1) up to 20% of normal meibum is formed of non-lipid components; (2) there are considerable differences between normal and DE meibum: the latter is enriched with non-lipid material, and forms lipid layers that are less structurally stable than those formed of healthy meibum. Based on those findings, we propose to assess the non-lipid content of mei- bum using: (a) metabolomic approaches based on HPLC-MS, GC-MS, and 2D-DESI MS; (b) Western blot with specific antibodies against major meibum and TF proteins; (c) histochemical approaches targeting proteins and polysaccharides; (d) various biophysical methods, such as hot stage polarized light microscopy, microcalori- metry, Brewster angle microscopy, to study rheological and melting characteristics of meibum and tear film lipids. We believe that our work will provide innovative information that may lead to new concepts in medical treatment of MG-related diseases and conditions, and may establish a link between them and dyslipidemia.

研究摘要 该项目的目标是阐明脂质生物合成的酶促途径， 由人类睑板腺 (HMG) 产生，统称为睑脂。 泪膜 (TF) 的重要组成部分，除其他功能外，还可以保护眼表免受 干燥的睑脂主要由非常长的链的独特混合物形成。 我们之前的项目致力于复杂脂质的生化和生物物理表征。 现在我们建议研究导致睑板腺脂质（ML）形成的生物合成途径。 提出了以下三方面的研究：（I）。 尽管对睑脂和 TF 的脂质组进行了表征，但对生物合成路径尚无明确的了解 ML 不寻常且通常独特的品质意味着它们是原位的。 和/或从头起源，这反过来又需要在 HMG 中实施适当的分子机制。 我们将研究主要脂质代谢酶和相关蛋白质的表达水平并绘制图谱，其中， 根据我们的初步研究，预计这将通过使用 HMG 来实现。 多种方法的组合，例如微阵列 RNA 分析、免疫组织化学/免疫细胞化学、 原位杂交、蛋白质组学和脂质组学 (II)。 包括胆固醇和 TAG，与 MG 功能障碍的较高发生率和/或严重程度相关。 据报道，ω3 脂肪酸 (ω3FA) 补充剂对人类和小鼠的眼表有积极影响。 然而，这些影响的原因和分子机制尚不清楚，我们将确定其平衡。 我们将测试 ML 的原位/从头生物合成与血脂的水平和成分之间是否存在 1)。 增加 ω3FA 药物剂量的摄入，以及 (2) 使用他汀类药物，可以对 睑脂的生物合成、成分和性质（III）最近，我们报道：（1）高达正常的20%。 睑脂是由非脂质成分组成；（2）正常和DE之间有相当大的差异。 睑脂：后者富含非脂质物质，并且脂质形成的层在结构上不太稳定 根据这些发现，我们建议评估睑脂的非脂质含量。 使用：(a) 基于 HPLC-MS、GC-MS 和 2D-​​DESI MS 的代谢组学方法；(b) 蛋白质印迹； 针对主要睑脂和 TF 蛋白的特异性抗体；(c) 针对蛋白质和 多糖；（d）各种生物物理方法，例如热台偏光显微镜、微热量 测量，布鲁斯特角显微镜，研究睑脂和泪膜的流变和熔化特性 我们相信我们的工作将提供可能带来医学新概念的创新信息。 治疗 MG 相关疾病和病症，并可能在它们与血脂异常之间建立联系。