Insights into the Lid Margin: Characterization of Membrane Associated Mucins in Ocular Surface Disease

项目摘要 这个项目的目标是为候选人提供必要的技能和培训，成为一个 眼表粘蛋白糖生物学领域的独立临床科学家。申请人将学习 分析高分子量O-糖基化眼粘蛋白的方法。分析这些 粘蛋白由于广泛的分支和许多聚糖附着而具有挑战性， 体内可获得的样本量有限。因此，申请人在这方面的培训 场将是非常有价值的和有益的眼表社区。眼部粘蛋白 表面在泪膜中发现，并附着在角膜和球结膜上， 润滑眼睛。结膜中的膜相关粘蛋白（MAM）形成糖萼， 对眼泪的亲水吸引力很重要。在干眼症（DED）中， 角膜和球结膜中粘蛋白的表达有助于其发病机制。而 眼表干燥被认为会导致眨眼时摩擦力增加， 眼睑内表面（睑结膜）的上皮细胞生理学上的干燥是 不清楚目标1中的实验将确定MAM MUC 1和MUC 16是否存在于细胞中。 睑结膜目的2中的实验将比较MUC 1的基因表达和蛋白水平， 和MUC 16在患有中度至重度DED的人的睑结膜中的表达。最后， 目的3中的实验将研究MUC 16对糖萼中半乳糖凝集素-3的亲和力。目标1和 2将使用真实的时间定量PCR分析来检测MUC 1和MUC 16的表达。细胞 将使用一种称为印迹细胞学的技术收集样本，然后进行基因处理。 表达和蛋白质定量。Aim 3将利用改进的狭缝印迹亲和测定来分析 MUC 16对半乳糖凝集素-3的亲和力，半乳糖凝集素-3是一种在糖萼中与MUC 16结合并有助于 它的屏障功能。为了实现这些目标，申请人组建了一个强大的指导团队。 主要导师Jason Nichols博士具有广泛的临床研究历史， 经验和分析专业知识。共同导师巴勃罗·阿圭索博士一直在研究眼部 表面粘蛋白超过十年，并知道这些分析所涉及的复杂性， 糖蛋白这项K23支持的研究将为未来的研究奠定基础。 干眼病的糖生物学基础和眼部炎症对粘蛋白的影响。

Project Summary The goal of this project is to provide the candidate with the necessary skills and training to become an independent clinician-scientist in the field of ocular surface mucin glycobiology. The applicant will learn methodologies to analyze high molecular weight O-glycosylated ocular mucins. Analysis of these mucins is challenging due to the extensive branching and numerous glycan attachments and is compounded by the limited sample size obtainable in vivo. Therefore, training of the applicant in this field would be highly valuable and beneficial to the ocular surface community. Mucins on the ocular surface are found both in the tear film and attached to the cornea and bulbar conjunctiva to primarily lubricate the eye. Membrane associated mucins (MAMs) in the conjunctiva form the glycocalyx which is important for hydrophilic attraction of the tears. In dry eye disease (DED), there are changes in the expression of mucins in the cornea and bulbar conjunctiva that contribute to its pathogenesis. While dryness on the ocular surface is thought to lead to increased friction during blinking, the impact of dryness on the epithelial cell physiology of the inner surfaces of the eyelid (palpebral conjunctiva) is unclear. Experiments in Aim 1 will determine whether MAMs MUC1 and MUC16 are present in the palpebral conjunctiva. Experiments in Aim 2 will compare gene expression and protein levels of MUC1 and MUC16 in the palpebral conjunctivas of humans with moderate to severe DED. Finally, experiments in Aim 3 will investigate the affinity of MUC16 for galectin-3 in the glycocalyx. Aims 1 and 2 will use real time quantitative PCR analysis to detect expression of MUC1 and MUC16. The cell samples will be collected using a technique known as impression cytology and then processed for gene expression and protein quantification. Aim 3 will utilize an adapted slot blot affinity assay to analyze the affinity of MUC16 for galectin-3, a lectin that associates with MUC16 in the glycocalyx and contributes to its barrier function. To accomplish these aims, the applicant has assembled a strong mentoring team. The primary mentor, Dr. Jason Nichols, has an extensive history of clinical research, mentoring experiences, and analytical expertise. The co-mentor, Dr. Pablo Argüeso, has been researching ocular surface mucins for over a decade and knows the intricacies involved with analysis of these glycoproteins. This K23 supported research will form the foundation for future research into the glycobiological basis of dry eye disease and the impact of ocular inflammation on mucins.