Understanding and Enhancing Repair of the Ear Drum

鼓膜（鼓膜）穿孔（TMP）是一种非常普遍的临床问题，尤其常见于幼儿。tmp可由身体创伤、压力变化以及最常见的急性中耳炎（AOM）引起，这是一种几乎所有儿童在三岁前都会患的疾病(1)。29.5%的AOM患儿出现TMPs，与既往OM病史相关的发病率增加(2)。虽然大多数tmp在2周内自行愈合，但大约6%的tmp不愈合并成为慢性疾病。在患者中，由于一系列因素，颞叶关节炎可能无法自发愈合。这个洞可能太大而无法修复(3)，或者可能形成上皮嵴阻止进一步愈合(4)。与AOM有关的感染因素也会抑制愈合。累及锤骨的穿孔也是有问题的(5)。慢性鼓膜炎会导致并发症，包括反复感染、中耳囊肿、耳痛、耳鸣和传导性听力损失，必须通过手术治疗（1型鼓膜成形术或鼓膜成形术）。本研究旨在揭示穿孔愈合的相关机制，以促进不能自然愈合的颞下颌关节的愈合。由于TM具有自发愈合的能力，有人认为膜必须包含其自身的干细胞/祖细胞群。一些研究小组利用表皮SC标记物6-整合素、B1 -整合素和细胞角蛋白19 （CK19）在人和大鼠TMs中寻找SC。这些标记物在茎柄、环周围、膜边缘和穿孔后呈阳性染色，这些标记物的表达显著增加[6]。Wnt信号是控制许多器官中干细胞/祖细胞活性的核心，并可能作为维持干细胞自我更新状态的生态位因子。此外，作为研究Wnt是否可能在TM中控制干细胞/祖细胞中发挥作用的初步研究的一部分，我们使用Axin2lacZ报告小鼠观察了Wnt在TM中的活性。响应Wnt活性的细胞位于TM的环区和柄周围，与其他假定的干细胞标记物的一般模式相匹配。因此，Wnt信号在细胞膜内的离散细胞群中是活跃的。塔克实验室已经开发了一种TMP方案，用于研究小鼠体内和外植体培养的耳膜穿孔，并收集了用于研究该问题的转基因模型。这项研究是由MRC资助的耳膜修复项目的博士后资助的。塔克实验室在中耳和外耳发育和修复方面具有特别的专业知识，并在该领域发表了许多论文。蒋教授是一名耳科医生，他将在治疗慢性颞叶炎患者方面提供专业知识。我们的目标是了解鼓膜在损伤过程中如何自我修复（涉及的细胞和信号通路），以及如何加强问题孔的愈合。体内平衡：哪些细胞有助于耳膜的更新？Wnt应答细胞（Axin2creERT2Tom标签）谱系追踪修复。什么细胞对损伤有反应？神经嵴，内胚层，中胚层，外胚层？操纵Wnt信号对修复的影响是如何的？过表达GOF小鼠，Axin2小鼠，功能丧失Wntless小鼠Cres: Pcagcre， K14ert2cre， Sox2ert2cre操纵信号通路如何影响体外修复：外植体培养的使用？对小鼠血管系统的影响

Tympanic membrane (ear drum) perforation (TMP) is a highly prevalent clinical problem, particularly common in young children. TMPs can result from physical trauma, pressure changes and, most commonly, from acute otitis media (AOM) a disease which almost all children will suffer from before the age of three (1). TMPs occur in 29.5% of children with AOM, with increased incidence associated with a previous history of OM (2). Although the majority of TMPs spontaneously heal within 2 weeks, approximately 6% do not heal and become chronic. In patients, TMPs can fail to spontaneously heal due to a range of factors. The hole may be too large to allow repair (3) or may form an epithelial ridge preventing further healing (4). Infectious factors, involved with AOM, also inhibit healing. Perforations involving the malleus also appear to be problematic (5). Chronic TMPs lead to complications including recurrent infections, middle ear cysts, otalgia, tinnitus and conductive hearing loss and have to be treated surgically (Type 1 Tympanoplasty or Myringoplasty). This proposal aims to uncover the mechanisms involved in perforation healing in order to enhance healing in TMPs that do not heal naturally. Due to the ability of the TM to spontaneously heal it has been suggested that the membrane must harbour its own stem/progenitor cell populations. Several groups have used the epidermal SC markers a6-integrin, B1 -integrin and cytokeratin 19 (CK19) to look for SCs in human and rat TMs. Positive staining for these markers was observed along the manubrium and around the annulus, at the edge of the membrane and after perforation a significant increase in expression of these markers was observed [6]. Wnt signalling is central to the control of stem/progenitor cell activity in a number of organs and may act as a niche factor to maintain stem cells in a self-renewing state. In addition, As part of a preliminary study to investigate whether Wnts may play a role in control of the stem/progenitor cells in the TM we looked at Wnt activity in the TM using the Axin2lacZ reporter mouse. Cells responding to Wnt activity were located in the annulus region of the TM and around the manubrium, matching the general pattern of other putative stem cell markers. Wnt signalling is therefore active in a discrete population of cells within the membrane. The Tucker lab has developed a TMP protocol for studying ear drum perforations in mice in vivo and in explant culture and has a collection of transgenic models for studying the problem. The research is supported by a postdoc on an MRC funded project grant on ear drum repair. The Tucker lab has particular expertise in middle and external ear development and repair and have published a number of papers in this area. Prof Jiang is an ear surgeon and will provide his expertise in dealing with chronic TMPs in patients.We aim to understand how the ear drum repairs itself during injury (the cells and signalling pathways involved) and how to enhance healing of problematic holes. Homeostasis: what cells contribute to turnover in the ear drum? Wnt responding cells (Axin2creERT2Tom label) Lineage tracing repair. What cells respond to injury? Neural crest, endoderm, mesoderm, ectoderm? Cres: Wnt1cre, Mesp1cre, Sox17icre, Sox2ert2cre, K14cre, K14ert2cre, Axin2ert2cre How does manipulating Wnt signalling impact on repair? Overexpresssion GOF mice, Axin2 mice, loss of function Wntless mice Cres: Pcagcre, K14ert2cre, Sox2ert2cre How does manipulating signalling pathways impact repair in vitro: use of explant cultures? Effect on vasculature Mesp1cretom mice Pharmacological impact on repair