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

项目摘要 该项目的目标是为候选人提供必要的技能和培训，使其成为 独立的临床医生-眼表粘蛋白糖生物学领域的科学家。申请者将了解到 分析高分子量O-糖基化眼粘蛋白的方法学。对这些问题的分析 粘蛋白是具有挑战性的，因为它具有广泛的分支和大量的葡聚糖连接 与体内可获得的有限样本量相结合。因此，对申请人进行这方面的培训 视野将是非常有价值的，对眼表社区有利。眼睛上的粘液 表面均可见泪膜，并附着于角膜和球结膜上，以 给眼睛涂点油。结膜中的膜相关粘蛋白(MAM)形成糖萼，它是 对于眼泪的亲水性吸引很重要。在干眼症(DED)中， 粘蛋白在角膜和球结膜中的表达与其发病机制有关。而当 眼睛表面的干燥被认为会导致眨眼期间摩擦增加，这种影响 眼睑内表面(眼睑结膜)上皮细胞生理学干燥 不清楚。目标1中的实验将确定MAM MUC1和MUC16是否存在于 眼睑结膜。AIM 2的实验将比较MUC1的基因表达和蛋白质水平 和MUC16在中、重度DED患者的眼睑结膜中的表达。最后， 目标3中的实验将研究MUC16与糖萼中Galectin-3的亲和力。目标1和 2将用实时定量聚合酶链式反应检测MUC1和MUC16的表达。单元格 样本将使用一种名为印迹细胞学的技术收集，然后进行基因处理 表达和蛋白质定量。AIM 3将利用一种适应的狭缝杂交亲和分析方法来分析 MUC16与Galectin-3的亲和力，Galectin-3是一种凝集素，在糖基化反应中与MUC16结合并对 它的屏障功能。为了实现这些目标，申请者组建了一支强大的指导团队。 主要导师Jason Nichols博士在临床研究、指导方面有丰富的历史 经验和分析专业知识。联合导师巴勃罗·阿古埃索博士一直在研究眼科 十多年的表面粘蛋白，并了解这些分析涉及的错综复杂的问题 糖蛋白。这项由K23支持的研究将为未来研究 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.