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

• 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
• 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

 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（由申请方提供）：肠粘膜上皮损伤损害屏障功能，可导致败血症和死亡。缺血和由此产生的缺氧在诸如新生儿坏死性小肠结肠炎、肠扭转、心肺疾病和出血性休克的疾病中显著地促进上皮损伤。肠上皮干细胞（IESC）由于其再生粘膜上皮屏障的能力而提供了有希望的治疗靶点。目前的证据支持存在两种IESC群体：a）隐窝基底柱状（CBC）细胞（Lgr 5富集）;和B）储备或更多的静止干细胞（QSC;集中在+4位置; Hopx富集;较慢的循环）。了解它们对上皮修复的贡献对于最佳治疗靶向至关重要。低氧诱导因子（hypoxia inducibe factor，HIF）通路调节细胞在低氧环境中的存活、增殖和分化。HIF途径激活Wnt-/β-连环蛋白途径，已知其对IESC存活、增殖和肠上皮止血至关重要。我们建议 猪缺血模型将有助于IESC研究向临床医学的转化和最终应用。该提案将研究不同IESC群体对缺血性损伤表现出不同抗性的假设，以及IESC中HIF途径激活介导这种抗性和/或缺血后IESC介导的严重损伤上皮的再生。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细胞是否显示出对缺血性损伤的不同抗性以及对后续修复和再生反应的不同贡献;和（2）缺血和后续再生期间IESC群体中HIF途径的作用。为了实现特定目标1，体内猪肠系膜血管闭塞模型将产生增加程度的缺血性损伤。CBC和QSC的生物标志物将用于定量组织学、蛋白质和mRNA分析，以确认缺血诱导的CBC损失但保留QSC的初步数据。两种IESC的特异性生物标志物与凋亡标志物表达的共定位将评估缺血对IESC死亡的影响。缺血和修复期间QSC相对于CBC增殖的时间过程使用与EdU、PCNA和磷酸化组蛋白3B的共定位来评估。缺血后和修复过程中HIF通路激活的时间过程将通过蛋白质和mRNA水平的介体测定来确定。在具体目标2中，将利用隐窝和细胞培养系统来操纵和定义HIF途径在响应缺氧中的功能作用。该项目的成功结果将确定缺血对IESC的影响，以及改善上皮修复的潜在新治疗靶点，并提供专门针对IESC和缺氧相关途径的细胞和分子生物学方面的额外专业知识。这些培训和职业发展活动将促进成功过渡到作为一名学术研究科学家的独立性。