Corneal Epithelial-Stromal Interactions During Regeneration and Fibrosis

项目摘要 细胞间通讯是角膜大部分功能的重要组成部分，包括纤维化， 再生和体内平衡。在很大程度上，细胞间的通讯被认为是由 释放大量可溶性生长因子和细胞因子以指导角膜创伤修复;然而， 根本的问题仍然是这些因素如何从一个细胞到相邻的细胞灌注任何特异性 cell.最近，对另一种细胞通信方法的兴趣激增。这种方法涉及 小泡的分泌和摄取，称为细胞外小泡（EV）。电动汽车可以长时间和短时间迁移 距离被其他细胞占据。电池之间的这种串扰是值得注意的，因为电动汽车携带“货物”。 由蛋白质、mRNA、miRNA和DNA组成，可以影响受体细胞中的许多功能。 事实上，最近的研究结果表明，癌症的发展涉及与电动汽车的串扰，其中癌细胞 产生作用于宿主细胞的EV，以产生促进癌细胞生长的微环境。 我们建议检查角膜伤口愈合是否涉及类似的机制，即上皮细胞释放 改变角膜细胞特性的EV。我们已经发现上皮EV可以刺激角膜细胞， 成为肌成纤维细胞，并且来自受伤细胞的上皮EV具有更大的作用。这里面有铅 我们假设上皮EV货物随着细胞迁移以愈合伤口而改变， 随后导致角膜细胞变成肌成纤维细胞，从而产生一个微环境， 促进他们的坚持。由于其清晰度和可及性，角膜提供了一个杰出的模型 以检查EV在体内或离体模型中通过基质的运动。将使用的技术包括EV 分离，蛋白质组学miRNA的分离和表征，透射电镜，共聚焦显微镜 显微镜和3D培养。与公共卫生的相关性-EV参与细胞间通讯， 几乎所有的生理和病理过程。因为它们有能力长途跋涉， 由于EV与特定细胞相互作用，因此EV被广泛检查用于递送治疗剂的有用性。我们将 确定EV局部应用于角膜是否可用作治疗方法。潜在地，任何 角膜损伤或疾病可以用EV治疗。

PROJECT SUMMARY Cell-cell communication is an important component of most functions of the cornea, including fibrosis, regeneration, and homeostasis. To a great extent, cell-cell communication has been thought to consist of the release of numerous soluble growth factors and cytokines to direct corneal wound repair; however an underlying question remains as to how these factors perfuse with any specificity from one cell to an adjacent cell. Recently, interest in another method of cell-communication has boomed. This method involves the secretion and uptake of small vesicles, termed Extracellular Vesicles (EVs). EVs can migrate long and short distances to be taken up by other cells. This crosstalk between cells is notable in that EVs carry “cargo” consisting of proteins, mRNA, miRNA, and DNA that can influence many of the functions in the recipient cell. Indeed, recent findings indicate that development of cancer involves crosstalk with EVs, where cancer cells produce EVs that act on the host's cells to produce a microenvironment that promotes growth of cancer cells. We propose to examine if corneal wound healing involves a similar mechanism, where epithelial cells release EVs that alter the properties of keratocytes. We have found that epithelial EVs can stimulate keratocytes to become myofibroblasts and that epithelial EVs from wounded cells have an even greater effect. This has lead us to the hypothesis that the epithelial EV cargo is altered as cells become migratory to heal a wound, subsequently causing the keratocyte to become myofibroblasts, which produce a microenvironment that promotes their persistence. Because of its clarity and accessibility, the cornea provides an outstanding model to examine movement of EVs through matrix in in vivo or ex vivo models. Techniques to be used include EV isolation, proteomics. miRNA isolation and characterization, transmission electron microscopy, confocal microscopy, and 3D cultures. Relevance to Public Health—EVs are involved in cell-cell communication in virtually all physiological and pathological processes. Because of their ability to travel long distances and interact with specific cells, EVs are widely being examined for usefulness to deliver therapeutics. We will determine if topical application of EVs to the cornea is useful as a therapeutic approach. Potentially, any corneal injury or disease could be treated with EVs.