Understanding the molecular mechanisms underlying tympanic membrane homeostasis and repair after injury

项目摘要/摘要 TM穿孔是一种非常常见的病理，患者表现为传导性听力。 损失。穿孔一般会在2-3周内自然愈合。虽然通常都是在没有 干预，每年进行75万次外科手术以闭合慢性穿孔，其中 伤口仍然敞开，原因不明。尽管患病率很高，但这种情况的原因尚不清楚。 对TM的基础生物学进行进一步的研究对于了解TM的伤口愈合是必要的 TM和慢性穿孔中的异常调节。 皮肤损伤，一种与TM相当的组织，已经被大量研究，但同样在... 需要对TM损伤的生物学进行深入调查。从我的导师最近的工作和 同事们，使用5-乙炔基-2‘-脱氧尿苷(EDU)标记，TM显示出特征的增殖 图案，主要集中在锤骨的手柄，第一个小骨嵌入TM，以及 环周围是TM的外环。角质形成细胞(KCs)从这些区域向下迁移并 径向向外，但损伤后的增殖情况尚不清楚。初步单细胞 RNA测序研究已经确定了动态平衡TM中的细胞群体通过转录 轮廓，揭示具有与其他上皮组织不同的特征的群体 像皮肤一样。这包括具有固有迁徙特性的基本KC种群。然而，这些是如何 已识别的细胞群体协调更新、分化和迁移是未知的，但它对于 理解这一点，就可以理解TM在这种情况下是如何保持动态平衡和自我修复的 受伤的原因。实验室的工作表明，在受伤时，TM能够识别伤害并产生 24小时内有强大的增殖反应。此外，这种扩散反应并不局限于 角质形成细胞层，也涉及间充质层和粘膜层。 鉴于这些初步研究，我的中心假设是TM的愈合涉及到 在动态平衡条件下不存在的亚型细胞的协调活动。在这份提案中， 我的第一个目标是定义穿孔后TM愈合的细胞动力学。对于我的第二个目标，我 将寻求了解骨形态发生蛋白(BMP)信号在维持细胞增殖中的作用 TM角质形成细胞处于动态平衡和损伤反应状态。除了我的导师和合作导师的 TM研究方面的专业知识，与Julie Sneddon、Max Krumel和Sarah Knox的合作支持 这项工作的可行性。这里提出的研究将旨在定义目前存在的特征细胞群 并确定它们的转录图谱。这将为TM如何 对受伤和治愈做出反应。此外，还将调查伤口愈合的潜在机制，以告知 TMS慢性穿孔的未来治疗。

PROJECT SUMMARY/ABSTRACT TM perforations are an extremely common pathology of which patients present with conductive hearing loss. Perforations, in general, spontaneously close within 2-3 weeks. Though commonly repaired without intervention, 750,000 surgical procedures are performed each year to close chronic perforations, in which the wound remains open for unknown reasons. Despite this high prevalence, the cause of this condition is unclear. A further investigation into the basic biology of the TM is compulsory to understanding the wound healing of the TM and what is dysregulated in chronic perforations. Wounding in skin, a tissue comparable to the TM, has been heavily studied, but the same sort of in- depth investigation into the biology of TM wounding is required. From recent work of my mentor and colleagues, using 5-ethynyl-2’-deoxyuridine (EdU) labeling, the TM displays a characteristic proliferation pattern, focused predominantly at the handle of the malleus, the first ossicle embedded into the TM, and around the annulus, the outer ring of the TM. From these regions, keratinocytes (KCs) then migrate down and radially outward, but what proliferation looks like in response to injury is unknown. Preliminary single-cell RNA sequencing studies have identified cell populations in the homeostatic TM via transcriptional profiles, revealing populations with characteristics distinct from those seen in other epithelial tissues like skin. This includes a basal KC population with inherent migratory properties. However, how these identified populations of cells coordinate renewal, differentiation, and migration is undefined, but it is critical to understand this to comprehend how the TM maintains homeostasis and repairs itself under the circumstance of injury. Work of the lab has shown that upon wounding, the TM is able to recognize injury and generate a robust proliferative response within 24 hours. Moreover, this proliferative response is not limited to the keratinocyte layer, but also involves the mesenchymal and mucosal layers. Given these Preliminary Studies, my central hypothesis is that healing of the TM involves the coordinated activities of subtypes of cells that are not present under homeostatic conditions. In this proposal, my first Aim is to define the cellular dynamics of healing in the TM following perforation. For my second Aim, I will look to understand the role of Bone Morphogenic Protein (BMP) signaling in maintaining the proliferation of TM keratinocytes under homeostasis and in response to injury. In addition to my mentor’s and co-mentor’s expertise with TM studies, collaborations with Julie Sneddon, Max Krummel, and Sarah Knox support the feasibility of this work. The studies proposed here will aim to define the characteristic cell populations present in the injured TM and define their transcriptional profiles. This will provide crucial insight into how the TM responds to injury and heals. Moreover, potential mechanisms for wound healing will be investigated to inform future therapies for chronic perforation of TMs.