Intestinal stem cell responses to ischemic injury in a large animal model

大型动物模型中肠道干细胞对缺血性损伤的反应

基本信息

批准号：
8967819
负责人：
Liara M Gonzalez
金额：
$ 12.16万
依托单位：
NORTH CAROLINA STATE UNIVERSITY RALEIGH
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-08-01 至 2020-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8967819
关键词：
Abdomen Abdominal Pain Address Affect Animal Model Apoptosis Biological Assay Biological Markers Biological Preservation Blood Vessels Cell Culture System Cell Cycle Cell Death Cell Hypoxia Cell Line Cell Survival Cells Cessation of life Chronic Clinical Clinical Medicine Columnar Cell Cultured Cells Data Development Diarrhea Disease Emergency Situation Environment Epithelial Epithelial Cells Epithelium Excision Experimental Models Family suidae Goals HIF1A gene Hemorrhagic Shock Hemostatic function Histologic Homeostasis Human Hypoxia Hypoxia-Inducible Factor Pathway In Vitro Injury Intestinal Volvulus Intestines Ischemia Ischemic Bowel Disease Literature Malnutrition Mediating Mediator of activation protein Mesenteric Vascular Occlusion Messenger RNA Modeling Molecular Molecular Biology Natural regeneration Necrotizing Enterocolitis Neonatal Operative Surgical Procedures Outcome Oxygen Pathway interactions Patients Phase Population Positioning Attribute Proteins Pulmonary Heart Disease Quality of life Recovery Regenerative response Research Resistance Risk Rodent Role Scientist Sepsis Short Bowel Syndrome Signal Transduction Pathway Stem Cell Research Stem cells Structure of intestinal gland System Testing Time Training Translations Villus Wit Work ascorbate base beta catenin career development caspase-3 clinically relevant crypt cell defined contribution functional outcomes improved in vivo injured lentiviral-mediated mortality new therapeutic target novel public health relevance repaired response self-renewal skills stem stem cell biology stem cell population therapeutic target translational approach

项目摘要

DESCRIPTION (provided by applicant): Intestinal mucosal epithelial injury compromises barrier function and can cause sepsis and death. Ischemia and the resulting hypoxia contribute significantly to epithelial damage in diseases such as neonatal necrotizing enterocolitis, volvulus, cardiopulmonary disease and hemorrhagic shock. Intestinal epithelial stem cells (IESCs) offer a promising therapeutic target due to their capacity to regenerate the mucosal epithelial barrier. Current evidence supports the existence of two IESC populations: a) Crypt Base Columnar (CBC) cells (Lgr5 enriched); and b) reserve or more quiescent stem cells (QSC; concentrated at the +4 position; Hopx enriched; slower cycling). Understanding their contribution to epithelial repair is critical to optimal therapeutic targeting. The hypoxia inducibe factor (HIF) pathway regulates cell survival, proliferation and differentiation in environments wit decreased oxygen. The HIF pathway activates the Wnt-/ß-catenin pathway which is known to be critical to IESC survival, proliferation and intestinal epithelial hemostasis. We propose that a porcine model of ischemia will aid in the translation and ultimate application of IESC research to clinical medicine. This proposal will investigate the hypothesis that distinct IESC populations wil show differing resistance to ischemic injury and that HIF pathway activation in IESCs mediates this resistance and/or IESC-mediated regeneration of severely injured epithelium after ischemia. Two specific aims will address this hypothesis by assessing: (1) if IESC cells expressing biomarkers of CBCs or QSCs show differential resistance to ischemic injury and different contributions to the subsequent repair and regenerative response; and (2) the role of the HIF pathway within the IESC populations during ischemia and subsequent regeneration. To accomplish specific aim 1, an in vivo porcine mesenteric vascular occlusion model will create increasing degrees of ischemic injury. Biomarkers of CBCs and QSCs will be used in quantitative histologic, protein and mRNA analyses to confirm preliminary data for ischemia-induced loss of CBCs but preservation of QSCs. Co-localization of the specific biomarkers of the two IESCs with expression of apoptosis markers will assess the impact of ischemia on IESC death. The time course of QSC versus CBC proliferation during ischemia and repair with be assessed using co- localization with EdU, PCNA and Phosphohistone 3B. The time course of HIF pathway activation after ischemia and during repair will be defined by assays of mediators of protein and mRNA levels. Crypt and cell culture systems will be utilized in specific aim 2 to manipulate and define the functional role of the HIF pathway in response to hypoxia. The successful outcome of this project will define the impact of ischemia on IESCs and potentially new therapeutic targets to improve epithelial repair as well as provide additional expertise in cel and molecular biology focused specifically on IESC and hypoxia-related pathways. These training and career development activities will promote a successful transition to independence as an academic research scientist.

描述（由申请人提供）：肠粘膜上皮损伤会损害屏障功能，并可能导致败血症和死亡，而由此引起的缺氧会导致新生儿坏死性小肠结肠炎、肠扭转、心肺疾病和失血性休克等疾病的上皮损伤。细胞（IESC）由于其再生能力而提供了一个有前途的治疗靶点目前的证据支持两个 IESC 群体的存在：a) 隐窝基底柱状 (CBC) 细胞（富含 Lgr5）；b) 储备或更多静止干细胞（QSC；集中在 +4 位置；富含 Hopx；了解它们对上皮修复的贡献对于缺氧诱导因子 (HIF) 通路在环境中调节细胞存活、增殖和分化至关重要。 HIF 通路激活 Wnt-/β-catenin 通路，这对于 IESC 的存活、增殖和肠上皮止血至关重要。猪缺血模型将有助于 IESC 研究的转化和最终应用到临床医学中。该提案将研究以下假设：不同的 IESC 群体将表现出不同的对缺血性损伤的抵抗力，并且 IESC 中的 HIF 通路激活介导了这种抵抗力和/或 IESC。缺血后严重受损上皮介导的再生有两个具体目标将通过评估来解决这一假设：(1)表达 CBC 或 QSC 生物标志物的 IESC 细胞是否表现出不同的耐药性。 (2) IESC 群体中 HIF 通路在缺血和随后再生过程中的作用。为了实现特定目标 1，将创建体内猪肠系膜血管闭塞模型。 CBC 和 QSC 的生物标志物将用于定量组织学、蛋白质和 mRNA 分析，以确认缺血引起的 CBC 损失但保留的初步数据。 QSC 与凋亡标记表达的两个 IESC 的特定生物标记的共定位将评估缺血对 IESC 死亡的影响。使用与 EdU 的共定位来评估缺血和修复过程中 QSC 与 CBC 增殖的时间过程。 PCNA 和磷酸组蛋白 3B。将利用 Crypt 和细胞培养系统来测定缺血后和修复期间的 HIF 通路激活的时间过程。具体目标 2 是确定 HIF 通路在缺氧反应中的功能作用。该项目的成功结果将确定操纵缺血对 IESC 的影响，以及改善上皮修复的潜在新治疗靶点，并提供细胞方面的额外专业知识。和分子生物学，特别关注 IESC 和缺氧相关途径。这些培训和职业发展活动将促进学术研究科学家成功过渡到独立。