Postnatal development of intestinal barrier repair

产后肠道屏障修复的发展

• 批准号: 10670758
• 负责人: Amanda Lee Ziegler
• 金额: $ 12.93万
• 依托单位: NORTH CAROLINA STATE UNIVERSITY RALEIGH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-15 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10670758
• 关键词: Adherent Culture; Adolescent; Affect; Annexins; Bacteria; Basement membrane; Basic Science; Biomedical Research; Biomimetics; Birth; Cell Culture Techniques; Cell Line; Cell physiology; Cells; Cellular Morphology; Clinical; Coculture Techniques; Collagen; Complex; Conditioned Culture Media; Data; Defect; Development; Disease; Enteral; Epidermal Growth Factor; Epithelial Cells; Epithelium; Experimental Designs; Failure; Family suidae; Focal Adhesion Kinase 1; Fostering; Foundations; Future; Genes; Goals; Health; Histology; Homeostasis; Hour; Human; Impairment; In Vitro; Infant; Injury; Institution; Intervention; Intestinal Diseases; Intestinal Volvulus; Intestines; Ischemia; Ischemic Bowel Disease; Knowledge; Lamina Propria; Literature; Mediating; Mediator; Medical; Mentorship; Modeling; Morbidity - disease rate; Mucous Membrane; Necrotizing Enterocolitis; Neonatal; Neuroglia; Outcome Study; PTK2 gene; Paracrine Communication; Pathway interactions; Patients; Phenotype; Play; Population; Productivity; Proteins; Recovery; Research; Research Personnel; Research Project Grants; Resources; Rodent; Role; Sagittaria; Scanning Electron Microscopy; Scientist; Sepsis; Signal Pathway; Signal Transduction; Small Intestines; Survival Rate; Techniques; Testing; Tissues; Toxin; Veterinary Medicine; Villus; Work; age related; career; cell motility; comparative; direct application; epithelial repair; experimental study; gastrointestinal; hydrogel scaffold; improved; in vitro Model; innovation; interest; intestinal barrier; intestinal epithelium; ischemic injury; jejunum; juvenile animal; mRNA Expression; mature animal; migration; mortality; neonatal patient; neonatal period; neonate; novel therapeutic intervention; paracrine; porcine model; postnatal; postnatal development; postnatal period; pro-EGF; programs; repaired; restoration; scaffold; skills; survival outcome; tool; treatment strategy; wound

Project Summary/ Abstract Intestinal diseases which involve ischemia such as volvulus or necrotizing enterocolitis cause mucosal barrier damage and are associated with poor survival in neonatal patients. The cause of higher mortality in neonates as compared to more mature patients has not been fully explained and represents a critical gap in our knowledge. Using a translational pig model of the human infant, I have found that while juvenile animals (6-weeks-old) repair rapidly by villus contraction and epithelial restitution, barrier repair is markedly impaired in neonates (2-weeks- old), characterized by a complete lack of epithelial restitution. Importantly, I found that the restitution defect in neonates can be rescued by the direct application of homogenized mucosal tissue from ischemia-injured small intestine from juvenile pigs. The mechanisms responsible for the age-dependent defect in restitution in neonates and the components of the juvenile mucosal tissues responsible for rescue of restitution are incompletely understood and form the basis of this proposal. Identifying how specific repair signaling mechanisms mature during the postnatal period has the potential to promote innovative discovery efforts leading to effective clinical interventions in neonatal patients with intestinal disease characterized by defective epithelial barrier function. A mixed population of subepithelial cells modulate epithelial cell functions in health and disease by secreting paracrine factors into the mucosal microenvironment. Of these cells, the subepithelial glial network is known to mature postnatally, and has been shown to directly regulate epithelial repair. I examined the glia within porcine jejunum and found an underdeveloped glial network in mucosa of neonates as compared to juveniles. Additionally, I found that epidermal growth factor and intracellular mediators of epithelial migration annexin A2 and focal adhesion kinase are reduced in neonatal mucosa. In order to study glial-epithelial interactions more closely, I will shift my studies to include advanced in vitro techniques. In a preliminary in vitro experiment, I found that media conditioned by glia cultured from juvenile pigs enhanced wound restitution in IPEC-J2 cells, a neonatal-derived intestinal epithelial cell line. These findings suggest glia may play an important role in postnatal development of barrier repair mechanisms. Therefore, I hypothesize that developmental glial factors within the subepithelial microenvironment signal restitution. To examine age-dependent differences in the effects of glia on epithelial repair, I will optimize and study primary enteric glial (aim 1) culture and primary intestinal epithelial culture on cutting-edge biomimetic collagen hydrogel scaffolds (aim 2) in the pig. As a young investigator completing these studies under new mentorship at a new top-ranking medical institution, I will expand my repertoire of skills in experimental design, execution and analysis to include basic science approaches to epithelial restitution mechanisms using advanced in vitro models. These studies will produce data to support new research grants (R03, R01) as I transition to research independence and provide the foundation for a productive career in comparative biomedical research to advance both human and veterinary medicine.

项目概要/摘要 涉及缺血的肠道疾病，如肠扭转或坏死性小肠结肠炎，会导致粘膜屏障 损害并与新生儿患者的不良生存有关。新生儿死亡率较高的原因 与更成熟的患者相比，尚未得到充分解释，这代表了我们知识中的一个关键差距。 使用人类婴儿的转化猪模型，我发现幼年动物（6周大）修复 由于绒毛收缩和上皮细胞迅速恢复，新生儿的屏障修复明显受损（2周- 老），其特征是完全缺乏上皮恢复。重要的是，我发现恢复缺陷 直接应用缺血损伤小儿的匀浆粘膜组织可挽救新生儿 来自幼猪的肠道。新生儿年龄依赖性恢复缺陷的机制 负责挽救恢复原状的幼年粘膜组织的成分不完全 理解并构成本提案的基础。确定特定修复信号机制如何成熟 产后期间有潜力促进创新发现工作，从而产生有效的临床效果 对患有以上皮屏障功能缺陷为特征的肠道疾病的新生儿患者进行干预。一个 上皮下细胞的混合群通过分泌调节上皮细胞在健康和疾病中的功能 旁分泌因子进入粘膜微环境。在这些细胞中，已知上皮下神经胶质网络 出生后成熟，并已被证明可以直接调节上皮修复。我检查了猪体内的神经胶质细胞 空肠，发现与青少年相比，新生儿粘膜中的神经胶质网络不发达。 此外，我发现表皮生长因子和上皮迁移膜联蛋白 A2 的细胞内介质 和粘着斑激酶在新生儿粘膜中减少。为了更多地研究胶质细胞与上皮细胞的相互作用 密切地，我将把我的研究转向包括先进的体外技术。在初步的体外实验中，我发现 由幼猪培养的神经胶质细胞调节的培养基增强了 IPEC-J2 细胞的伤口恢复， 新生儿来源的肠上皮细胞系。这些发现表明神经胶质细胞可能在产后发​​挥重要作用 障碍修复机制的发展。因此，我假设发育神经胶质因子 上皮下微环境信号恢复。研究神经胶质细胞对年龄的影响存在差异 上皮修复，我将优化和研究原代肠胶质细胞（目标1）培养和原代肠上皮 在猪的尖端仿生胶原水凝胶支架上进行培养（目标 2）。作为一名年轻的调查员 在新的顶级医疗机构的新指导下完成这些研究，我将扩展我的 实验设计、执行和分析方面的技能，包括基本科学方法 使用先进的体外模型研究上皮恢复机制。这些研究将产生数据来支持 新的研究补助金（R03，R01），因为我过渡到研究独立性并为 在比较生物医学研究方面取得富有成效的职业生涯，以促进人类和兽医医学的发展。