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

研究摘要 本项目的目标是阐明脂质生物合成的酶途径， 由人睑板腺（HMG）产生，统称为睑脂。Meibum是一个 泪液膜（TF）的一个至关重要的部分，除了其他功能外，它还保护眼表面免受 干燥睑脂主要是由一种独特的混合物，非常，非常长的链， 复合脂质我们以前的项目致力于生物化学和生物物理表征， 睑板脂质（ML）。现在，我们打算研究导致 HMG中的睑脂。本文提出了以下三条研究思路：（1）.尽管最近取得了进展， 虽然对睑脂和TF的脂质体的表征没有明确的认识，但对生物合成途径没有明确的认识- 参与睑脂生物合成的途径。ML的不寻常的，通常是独特的品质意味着它们的原位 和/或从头起源，这又需要在HMG中实施适当的分子机制。 我们将研究表达水平，并绘制主要的脂质代谢酶和相关蛋白质， 根据我们的初步研究，预计会出现在HMG中。这将通过使用 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。（二）.最近的报告表明，高总血脂水平， 包括胆固醇和TAG与MG功能障碍的更高发生率和/或严重性相关。此外， 据报道，ω3脂肪酸（ω3FA）补充剂对人和小鼠的眼表面具有积极影响。 然而，这些影响的原因和分子机制尚不清楚。我们将决定余额 ML的原位/从头生物合成与血脂水平和组成之间的关系。我们将测试如果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、组成和性质。（三）。最近，我们报道：（1）高达正常的20% 睑脂由非脂质成分组成;（2）正常与DE之间存在显著差异 睑脂：后者富含非脂质物质，并形成结构稳定性低于 那些由健康的睑脂形成的。基于这些发现，我们建议评估非脂质含量的梅- 使用：（a）基于HPLC-MS、GC-MS和2D-DESI MS的代谢组学方法;（B）Western印迹， 针对主要睑脂和TF蛋白的特异性抗体;（c）靶向蛋白质的组织化学方法， （d）各种生物物理方法，例如热台偏振光显微镜、微热-热分析法、热分析法等。 方法：采用光学显微镜、布鲁斯特角显微镜、透射电镜、 脂质。我们相信，我们的工作将提供创新的信息，可能导致新的概念，在医疗 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。

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.