Enteric Nervous System Development and Function in Human Intestinal Organoids

人肠类器官中肠神经系统的发育和功能

• 批准号: 10525822
• 负责人: Allison Leigh Speer
• 金额: $ 15.5万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-11 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10525822
• 关键词: Enteric; Nervous; System; Development; Function

This application outlines a proposal to advance the research career of a promising young investigator within a mentored setting. Successful completion will enable the candidate to establish a career as an independent NIH-funded surgeon-scientist, conducting translational research focused on identifying the processes required to generate functional tissue-engineered intestine as a cure for pediatric intestinal failure. Background: Intestinal failure is a survivable but morbid syndrome that afflicts tens of thousands of children. Contemporary management is inadequate and costly. Tissue-engineered intestine is a potential solution; however, complex intestinal functions have yet to be confirmed in existing models. Thus, understanding the processes required to generate a functional intestine are a critical unmet need and a high priority for the NIH. The enteric nervous system (ENS) is crucial for intestinal functions including barrier maintenance and peristalsis. Serotonin receptors (e.g. 5-HT4R), expressed by intestinal epithelial cells and enteric neurons, promote neurogenesis and regulate intestinal barrier function and motility. Based on preliminary data and published work, we hypothesize that human intestinal organoid (HIO) function depends on the ENS, and 5-HT4R is essential for proper ENS development and function within HIOs. Research Design and Methods: Three aims will test this hypothesis in HIOs in vitro and transplanted HIOs (tHIOs) in vivo± ENS. We will employ gain and loss of function experiments using 5-HT4R genetic knockouts and pharmacological 5-HT4R agonists/antagonists. Aim 1 will evaluate the role of 5-HT4R during ENS development in HIOs utilizing 3-D immunofluorescent (IF) advanced imaging as well as RT-qPCR to locate and quantify the ENS. Aim 2 will assess if 5-HT4R is required for barrier function in tHIOs by measuring tight junction production and morphology with IF staining, Western Blot, Transmission Electron Microscopy, and RTqPCR as well as determining transepithelial resistance and permeability in an Ussing Chamber ex vivo. Aim 3 will ascertain if 5-HT4R is indispensable for peristalsis in tHIOs using non-invasive, dynamic imaging in vivo and measuring contractility ex vivo. This project is novel because no prior study has examined the role of the ENS in barrier function in HIOs nor applied these new imaging methods. This project is novel and innovative in proposing a mechanism by which ENS development and function in HIOs are regulated by 5-HT4R. Career Development Plan & Goals: Over this 5 year award, the principal investigator will have 75% protected time to expand her background in intestinal tissue-engineering to gain new skills in ENS development and neurogastroenterology. Completion will propel the field forward and launch her independent research career. Research Environment: The proposed research will be carried out at UTHealth within the Texas Medical Center, the world's largest medical center which seeks to nurture cross-institutional collaboration, creativity, and innovation. This environment has an established success of nurturing the careers of junior investigators.

该应用程序概述了推进Promise Young的研究职业的建议 在设置问题的问题上。成功完成将使候选人能够建立职业 作为一名独立的NIH资助的外科医生科学家，进行转化研究的重点是确定 产生功能性组织工程肠所需的过程是治愈小儿肠道衰竭的方法。 背景：肠道衰竭是一种可生存但莫比的综合症，折磨了数万个 孩子们。当代管理不足和昂贵。组织工程肠是一种潜力 解决方案;但是，在现有模型中尚未确认复杂的肠道功能。那， 了解生成功能性肠所需的过程是至关重要的未满足需求和高度 NIH的优先级。肠神经系统（ENS）对于包括障碍在内的肠道功能至关重要 维护和蠕动。 5-羟色胺受体（例如5-HT4R），由肠上皮细胞和 肠神经元，促进神经发生并调节肠道屏障功能和运动。基于 初步数据和已发表的工作，我们假设人类肠癌（HIO）功能取决于 在ENS和5-HT4R上，对于HIO内的ENS开发和功能至关重要。 研究设计和方法：三个目标将在体外和移植的HIOS中检验此假设 （Thios）体内。我们将使用5-HT4R遗传基因敲除实验的增益和丧失实验 和药物5-HT4R激动剂/拮抗剂。 AIM 1将评估ENS期间5-HT4R的作用 使用3-D免疫荧光（IF）高级成像以及RT-QPCR的HIO开发 量化ENS。 AIM 2将通过测量紧密来评估THIOS屏障功能是否需要5-HT4R 结合染色，蛋白质印迹，透射电子显微镜和RTQPCR的连接生产和形态 以及确定室内室内的换皮性和渗透性。目标3 将确定使用非侵入性的动态成像在体内使用5-HT4R是否对于蠕动中的蠕动是否是必不可少的 并测量离体收缩力。该项目是新颖的，因为没有先前的研究检查了 在HIO中的屏障功能中的ENS或应用这些新成像方法。这个项目是新颖而创新的 提出了一种机制，该机制通过5-HT4R调节HIO中的ENS开发和功能。 职业发展计划和目标：在这5年奖上，首席调查员将受到75％的保护 是时候扩大她在肠道组织工程方面的背景，以获得ENS开发和 神经胃肠病学。完成将推动该领域的前进并启动她的独立研究生涯。 研究环境：拟议的研究将在德克萨斯州医学的UTHealth进行 中心是世界上最大的医疗中心，该中心旨在护理跨机构合作，创造力， 和创新。这种环境在培养初级调查人员的职业方面取得了良好的成功。