Post-natal development of enteric glial cell-epithelial interactions in repair of ischemic-injured intestine

项目摘要/摘要 如果不能迅速恢复上皮覆盖，肠粘膜屏障的破坏会导致败血症和死亡， 尤其是在新生儿中。与更成熟的人相比，婴儿死亡率更高的原因是 肠道损伤尚未得到解释。婴儿死亡率高是由与缺血/相关的疾病引起的 再灌注(I/R)损伤，包括坏死性小肠结肠炎和肠扭转。在幼猪(6-8周龄)中，我们 广泛研究了以上皮细胞迁移(恢复)为标志的显著快速的肠道修复。 然而，在这项提案的修订版中，我们最近发表的一项研究表明， 令人感兴趣的年龄依赖性缺陷在缺血性损伤后粘膜修复的恢复中几乎全部 新生仔猪缺乏恢复能力。有趣的是，修复中的缺陷可以通过一种匀浆的 缺血损伤的幼体匀浆，提示更多的成熟细胞群刺激修复。一个细胞 特别令人感兴趣的群体是肠神经胶质细胞(EGC)网络，我们已经显示出它的成熟 出生后在猪身上。在修订后的提案中，我们现在已经表明，猪EGC条件化媒体 刺激受损新生猪IPEC-J2细胞的细胞迁移(恢复)。然而，在这方面仍然存在差距 我们对上皮细胞和潜在的EGC之间的信号机制的了解导致 赔偿。为了解决这一差距，我们对缺血损伤的新生儿和 青少年粘膜，并创建了一种公正的发现管道方法，专注于显著不足的 新生儿体内的信号通路。一种这样的途径包括EGF，它由EGC分泌为 原表皮生长因子和上皮细胞膜联蛋白A2，通过信号转导β1-整合素的内在化来启动细胞迁移。我们的 中心假说是缺血损伤粘膜的修复依赖于EGC的出生后迁移 进入固有层，以便发出损伤上皮有效修复的信号。我们将对此进行测试 有两个特定目的的假说：1)确定年龄相关的迁移信号机制缺陷 伤口附近的肠上皮细胞。我们的工作假设是，有一个年龄相关的 损伤后上皮修复的发展，至少部分是通过膜联蛋白A2信号。2)确定 围产期粘膜EGC网络结构、密度和分泌体的变化。我们的工作假设是 上皮附近没有足够的EGCs分泌促进屏障的因子，包括原EGF。 以刺激新生儿恢复健康。为了检验这些特定的目标，我们将使用我们独特的年龄相关 猪粘膜修复模型、猪新生猪IPEC-J2细胞损伤模型、猪原代培养 EGC，EGC通过质谱学的分泌组分析，以及最新的神经胶质细胞成像技术， 包括启用免疫标记的溶剂清除器官的三维成像(IDISCO)。使用 不偏不倚的发现管道方法，我们期望能够定义有缺陷的信令机制 在新生儿受损的肠道中，最终可以靶向提高肠衰竭患者的存活率

PROJECT SUMMARY/ ABSTRACT Breaches in the intestinal mucosal barrier lead to sepsis and death if epithelial coverage is not rapidly restored, particularly in neonates. The reason for higher mortality in infants as compared to more mature individuals with intestinal injury has not been explained. High infant mortality results from diseases associated with ischemia/ reperfusion (I/R) injury, including necrotizing enterocolitis and volvulus. In juvenile pigs (6-8-weeks of age), we have extensively studied the remarkably rapid intestinal repair marked by epithelial cell migration (restitution). However, in a recently published study featured in the revised version of this proposal, we have shown an intriguing age-dependent deficiency in the recovery of mucosal repair following ischemic injury with a near-total lack of restitution in neonatal piglets. Interestingly, this defect in repair can be rescued by a homogenate of ischemic injured juvenile homogenate, suggesting more mature cell populations stimulate restitution. One cell population of particular interest is the enteric glial cell (EGC) network, which we have shown matures postnatally in pigs. In the revised proposal, we have now shown that porcine EGC-conditioned media stimulates cell migration (restitution) in wounded neonatal porcine IPEC-J2 cells. However, a gap remains in our knowledge regarding the signaling mechanisms between epithelium and underlying EGC that results in restitution. To address this gap, we have performed RNAseq analyses of ischemic-injured neonatal and juvenile mucosa, and created an unbiased discovery pipeline approach to focus on significantly deficient signaling pathways in the neonate. One such pathway includes EGF, which is secreted by the EGC as proEGF, and epithelial annexin A2, which initiates cell migration by signaling internalization of β1-integrin. Our central hypothesis is that repair of ischemic-injured mucosa is reliant on postnatal migration of EGC into the lamina propria in order to signal wounded epithelium to efficiently restitute. We will test this hypothesis with two specific aims: 1) Determine age-dependent defects in migration signaling mechanisms in wound-adjacent intestinal epithelial cells. Our working hypothesis is that there is an age-dependent development of epithelial restitution in response to injury at least in part via annexin A2 signaling. 2) Determine perinatal changes in mucosal EGC network structure, density and secretome. Our working hypothesis is that there are insufficient EGCs secreting barrier-promoting factors, including proEGF, in proximity to the epithelium to stimulate restitution in neonates. To examine these specific aims, we will use our unique age-dependent porcine model of mucosal repair, a wounded porcine neonatal IPEC-J2 cell model, primary culture of porcine EGC, EGC secretome analyses via mass spectrometry, and state of the art imaging techniques for glial cells, including immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO). Using an unbiased discovery pipeline approach, we expect to be able to define signaling mechanisms that are deficient in neonatal injured intestine that can ultimately be targeted to enhance survival of patients with intestinal failure