Enteric Nervous System Development and Function in Human Intestinal Organoids

这份申请概述了一项建议，以促进有前途的年轻人的研究事业 指导环境中的调查员。成功完成工作将使应聘者能够建立自己的职业生涯 作为一名由NIH资助的独立外科医生兼科学家，进行翻译研究的重点是确定 用于治疗小儿肠衰竭的功能性组织工程化肠道所需的过程。 背景：肠衰竭是一种可存活但病态的综合征，它困扰着成千上万的 孩子们。现代管理既不充分，也代价高昂。组织工程化肠道是一种潜在的 然而，复杂的肠道功能尚未在现有的模型中得到证实。因此， 了解产生有功能的肠道所需的过程是一个关键的未得到满足的需求和高要求 美国国立卫生研究院的优先事项。肠道神经系统(Ens)对包括屏障在内的肠道功能至关重要。 维护和蠕动。5-羟色胺受体(如5-HT4R)，由肠上皮细胞和 肠道神经元，促进神经发生，调节肠道屏障功能和运动。基于 初步数据和已发表的工作，我们假设人类肠道器官(HIO)的功能取决于 在ENS上，5-HT4R对ENS的正常发育和HIO内的功能是必不可少的。 研究设计和方法：三个目标将在体外和移植的HIO中验证这一假说 (硫代)在体内。我们将使用5-HT4R基因敲除进行功能得失实验 和药理上的5-HT4R激动剂/拮抗剂。目标1将评估5-HT4R在ENS中的作用 应用三维免疫荧光(IF)先进成像和RT-qPCR定位和检测HIO的研究进展 量化ENS。Aim 2将通过测量Strong来评估5-HT4R是否是THOS屏障功能所必需的 用IF染色、Western Blot、透射电子显微镜和RTqPCR观察接头的产生和形态 以及在体外Ussing小室中测定跨上皮阻力和渗透性。目标3 将使用非侵入性的体内动态成像来确定5-HT4R是否是THOS蠕动所必需的 以及测量体外的收缩能力。这个项目是新颖的，因为之前没有任何研究考察过 这些新的成像方法也没有应用于HIOS的屏障功能。该项目在以下方面具有创新性 提出了5-HT4R调控ENS在HIO中发育和功能的机制。 职业发展计划和目标：在这5年的奖励中，首席调查员将获得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。完成后将推动该领域向前发展，并开始她的独立研究生涯。 研究环境：拟议中的研究将在德克萨斯大学卫生分校的德克萨斯医疗中心进行 该中心是世界上最大的医疗中心，致力于培养跨机构合作、创造力、 和创新。这种环境在培养初级调查人员的职业生涯方面取得了既定的成功。

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.