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（由适用提供）：肠粘膜上皮损伤损害了障碍功能，并可能导致败血症和死亡。缺血和由此产生的缺氧对诸如新生儿坏死性小肠结肠炎，沃尔图鲁斯，心肺疾病和出血性休克等疾病的上皮损害显着促进。肠上皮干细胞（IESC）由于能力再生粘膜上皮屏障而提供了有前途的治疗靶标。当前的证据支持两个IESC种群的存在：a）加密基柱（CBC）细胞（LGR5富集）； b）储备或更多静止的干细胞（QSC；集中在+4位置；富含Hopx；较慢的骑行）。了解它们对上皮修复的贡献对于最佳治疗靶向至关重要。缺氧诱导因子（HIF）途径调节环境中的细胞存活，增殖和分化降低氧气。 HIF途径激活Wnt-/ß-catenin途径，该途径对IESC存活，增殖和肠上皮止血至关重要。我们建议 缺血的猪模型将有助于将IESC研究的翻译和最终应用于临床医学。该提议将调查以下假设：不同的IESC种群将显示出对缺血性损伤的不同耐药性，而IESCS中的HIF途径激活了这种耐药性和/或IESC介导的缺血上皮细胞的再生。通过评估：（1）表达CBCS或QSC的IESC细胞表现出对缺血性损伤的不同耐药性以及对随后的修复和再生反应的不同贡献，将解决这一假设的两个具体目标：（1） （2）在缺血和随后再生期间，HIF途径在IESC种群中的作用。为了完成特定的目标1，体内猪肠系膜血管闭塞模型将造成越来越多的缺血性损伤。 CBC和QSC的生物标志物将用于定量的组织学，蛋白质和mRNA分析，以确认缺血诱导的CBC丢失但保留QSC的初步数据。两种IESC的特定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。缺血期间QSC与CBC增殖的时间过程，并使用与EDU，PCNA和磷酸组织3B共定位进行评估。缺血后和修复过程中HIF途径激活的时间过程将由蛋白质和mRNA水平的介体的主张来定义。地穴和细胞培养系统将用于特定目标2中，以操纵和定义HIF途径对缺氧的功能作用。该项目的成功结果将定义缺血对IESC和潜在的新治疗靶标的影响，以改善上皮修复，并在CEL和分子生物学方面提供更多专门针对IESC和缺氧相关途径的专业知识。这些培训和职业发展活动将促进作为学术研究科学家的成功过渡到独立性的过渡。