Understanding Skin Tissue Repair in Live Mammals

了解活体哺乳动物的皮肤组织修复

• 批准号: 10677810
• 负责人: Valentina Greco
• 金额: $ 69.93万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-03-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10677810
• 关键词: Address; Affect; Age; Behavior; Behavioral; Cells; Cellular biology; Cessation of life; Collagen; Communication; Complex; Data; Deposition; Dermis; Disease; Ear; Ensure; Environment; Epidermis; Epithelial Cells; Epithelium; Equilibrium; Exhibits; Extracellular Matrix; Failure; Fibroblasts; Foundations; Gap Junctions; Genetic; Genetic Diseases; Goals; Growth; Health; Homeostasis; Hour; Human; Imaging technology; Individual; Injury; Intestines; Knowledge; Label; Laboratories; Malignant Neoplasms; Mammals; Mediating; Membrane; Microscopy; Modeling; Mosaicism; Mus; Mutation; Oncogenic; Organ; Patients; Physiological; Population; Prevention; Process; Proliferating; Research; Resolution; Role; Signal Pathway; Signal Transduction; Skin; Skin Tissue; Skin repair; Somatic Mutation; Squamous cell carcinoma; Structure; Testing; Therapeutic; Time; Tissues; Visualization; Work; aged; cell behavior; cell type; chronic wound; clinically relevant; coping; epidermal stem cell; epithelial repair; epithelial stem cell; experimental study; fitness; flexibility; genetic manipulation; healing; imaging approach; improved; in vivo; injury and repair; innovation; intravital imaging; intravital microscopy; mosaic; mouse model; mutant; novel; pharmacologic; reconstruction; repaired; response; self-renewal; single cell sequencing; spatiotemporal; tissue repair; tumor; wound closure; wound healing

Skin protects our body against the environment, and its ability to repair upon injury is directly connected to both disease and survival. Failure to properly repair injured tissue can result in chronic wounds, which are associated with severe complications and even death. Multiple cell types, like epithelial cells and fibroblasts, must coordinate their behaviors to achieve injury repair. These processes have so far mainly been studied in genetically homogeneous mouse models. However, healthy skin contains many clones harboring somatic mutations, including oncogenic Ras mutations which are the leading cause of Squamous Cell Carcinoma. How genetically diverse epithelial cells affect injury repair is unknown. The goal of this proposal is to unravel how cell behaviors are properly orchestrated on the single-cell and tissue-scale level during repair. The critical barrier to addressing these fundamental questions lies in the inability to study these dynamic processes in an intact mammal. To this end, my laboratory has established an in vivo strategy to directly visualize and manipulate epithelial cells and fibroblasts in the skin of live mice. We have previously used this strategy to define the roles of epithelial cells and fibroblasts in homeostasis, and the complex spatiotemporal organization of epithelial cell behaviors during repair. We hypothesize that epithelial cells and fibroblasts use flexible behaviors to enable proper wound healing of genetically mosaic skin. We will first define how a mosaic epithelium responds to injury (Aim 1). Our preliminary results show that a mosaic Hras mutant skin epithelium heals at a normal rate and does not induce tumors, but that WT and Hras mutant cells exhibit different behaviors during repair. We hypothesize that the behavioral flexibility of WT cells confers the ability to contain their Hras mutant neighbors and ultimately achieve normal injury repair. To test this, we will define the epithelial cell behaviors and evaluate the roles of signaling pathways during injury repair of an Hras-mosaic skin epithelium. We will then examine the roles of fibroblasts in contact with epithelial cells during injury repair (Aim 2). We hypothesize that direct communication between fibroblasts in the upper dermis and epithelial cells in the epidermis coordinates cell behaviors and repair. To test this, we will define the behaviors and functions of fibroblasts in the presence of WT epithelial cells and an Hras-mosaic epithelium. We will achieve both these aims by combining intravital microscopy with genetic and pharmacological manipulations of distinct repair behaviors or resident cell types in vivo. The proposed experiments will allow us to dissect the coordination and functional significance of distinct cell activities, populations, and interactions during repair using an integrated approach of cutting-edge imaging technology, genetic manipulation, cell biology, and single cell sequencing. Given that many aspects of injury repair are widely conserved in other organs, our findings will be relevant to other tissues as well, and will provide an important foundation to improve injury repair in a variety of patients.

皮肤保护我们的身体免受环境的影响，它在受伤后的修复能力直接与 对疾病和生存的影响。不能正确修复受伤的组织会导致慢性伤口， 伴随着严重的并发症甚至死亡多种细胞类型，如上皮细胞和成纤维细胞， 必须协调他们的行为以实现损伤修复。迄今为止，这些过程主要是在 遗传同质小鼠模型。然而，健康的皮肤包含许多克隆窝藏体细胞 突变，包括致癌Ras突变，这是鳞状细胞癌的主要原因。 遗传多样性上皮细胞如何影响损伤修复尚不清楚。本提案的目的是 揭示修复过程中细胞行为如何在单细胞和组织规模水平上正确协调。的 解决这些基本问题的关键障碍在于无法研究这些动态过程 在一个完整的哺乳动物。为此，我的实验室已经建立了一种体内策略，直接可视化， 操纵活小鼠皮肤中的上皮细胞和成纤维细胞。我们以前用这个策略来定义 上皮细胞和成纤维细胞在体内平衡中的作用，以及上皮细胞和成纤维细胞的复杂时空组织。 修复过程中的上皮细胞行为。我们假设上皮细胞和成纤维细胞使用灵活的行为 以使基因嵌合皮肤的伤口能够适当愈合。 我们将首先定义镶嵌上皮对损伤的反应（目的1）。我们的初步结果显示， 嵌合型Hras突变的皮肤上皮以正常速度愈合，不诱发肿瘤，但WT和Hras突变的皮肤上皮以正常速度愈合， 突变细胞在修复过程中表现出不同的行为。我们假设WT细胞的行为灵活性 赋予其包含Hras突变邻居并最终实现正常损伤修复的能力。为了验证这个， 我们将定义上皮细胞的行为，并评估信号通路在损伤修复过程中的作用。 Hras-镶嵌皮肤上皮。然后，我们将研究成纤维细胞在与上皮细胞接触过程中的作用。 损伤修复（目标2）。我们推测，真皮上层成纤维细胞之间的直接通讯， 表皮中的上皮细胞协调细胞行为和修复。为了测试这一点，我们将定义 以及在WT上皮细胞和Hras镶嵌上皮存在下成纤维细胞的功能。我们将实现 这两个目的都是通过将活体显微镜与不同的遗传和药理学操作相结合， 修复行为或体内驻留细胞类型。 拟议的实验将使我们能够剖析协调和功能的意义， 不同的细胞活动，种群，以及修复过程中的相互作用， 成像技术、遗传操作、细胞生物学和单细胞测序。鉴于许多方面的 损伤修复在其他器官中广泛存在，我们的发现也将与其他组织相关， 为改善各种患者的损伤修复提供了重要基础。