Ibuprofen and enteric neural crest precursor migration

布洛芬和肠神经嵴前体迁移

• 批准号: 8917209
• 负责人: Ellen Merrick Schill
• 金额: $ 4.81万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-20 至 2016-09-19
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8917209
• 关键词: Abdomen; Actins; Adhesions; Affect; Age; Age-Years; Anti-Inflammatory Agents; Anti-inflammatory; Biochemical; Cause of Death; Cell Survival; Cells; Cessation of life; Child; Colon; Congenital Abnormality; Congenital Megacolon; Cytoskeleton; Data; Defect; Development; Diet; Disease; Distal; Distant; Enteral; Enteric Nervous System; Environmental Exposure; Environmental Risk Factor; Enzymes; Exhibits; Exposure to; Family; Fetal Development; First Pregnancy Trimester; Fishes; Focal Adhesions; Folic Acid; Genes; Genetic Predisposition to Disease; Genetic Risk; Goals; Health; Human; Ibuprofen; Image; Immunohistochemistry; In Vitro; Incidence; Individual; Infant; Intestines; Investigation; Lead; Length; Life; Measures; Mediating; Medicine; Microscopy; Molecular; Morbidity - disease rate; Mus; Mutation; Nervous system structure; Neural Crest; Neural Tube Defects; Neuroglia; Neurons; Obstruction; PTGS1 gene; PTGS2 gene; Pathway interactions; Pharmaceutical Preparations; Physiology; Population; Preclinical Drug Evaluation; Pregnancy; Proteins; Regulation; Research Project Grants; Risk; Severities; Structure; Supplementation; Susceptibility Gene; Testing; Therapeutic; Time; Transgenic Animals; United States; Vertebrates; Vomiting; Woman; Zebrafish; base; cell motility; cellular imaging; critical period; cyclooxygenase 1; disease-causing mutation; disorder risk; effective intervention; gene environment interaction; gene interaction; improved; in utero; in vivo; inhibitor/antagonist; migration; mortality; mouse model; nervous system development; novel; null mutation; premature; prevent; research study; time use

DESCRIPTION (provided by applicant): Birth defects are the most common cause of death in children under one year of age in the United States. The best way to reduce the morbidity and mortality of specific birth defects is to develop strategies to prevent them from occurring, as exemplified by the decrease of neural tube defects due to folic acid supplementation. Hirschsprung Disease (HSCR) is a birth defect of the enteric nervous system that occurs when enteric neural crest-derived cells (ENCCs) do not migrate all the way to the end of the bowel. The resulting distal bowel aganglionosis leads to a functional obstruction, which causes vomiting, abdominal distension and predisposes to premature death. There are many proteins important for normal ENCC migration and mutations in these genes increase the risk of HSCR, but none of these mutations cause disease in all affected children. Therefore, children who have HSCR must have additional factors that further increase their HSCR risk. To develop strategies to decrease HSCR occurrence, it is important to identify alterable factors, such as maternal diet or medications that could in combination with genetic defects cause disease in a child who would otherwise have been born healthy. Ibuprofen, a non-steroidal anti-inflammatory drug (NSAID) commonly used in pregnancy, inhibited ENCC migration in a zebrafish drug screen, causing a HSCR-like distal bowel aganglionosis in affected fish. Experiments in mammalian primary culture confirmed that ibuprofen slows ENCC migration and appears to disrupt actin cytoskeletal and focal adhesion regulation needed for efficient migration. The goals of this proposal are to determine the biochemical mechanism of ibuprofen's inhibition of ENCC migration and to determine if ibuprofen increases the likelihood of HSCR in genetically predisposed individuals. The biochemical mechanisms that ibuprofen disrupts to inhibit ENCC migration will be investigated using cutting edge time-lapse live cell imaging of fluorescently-tagged proteins. These investigations will allow for in depth analysis of ENCC actin protrusions and the dynamics of focal adhesions. In addition, ENCCs from transgenic animals with null mutations in Cox1 and Cox2, enzymes inhibited by ibuprofen, will be analyzed by immunohistochemistry and time-lapse imaging to determine if ibuprofen inhibits ENCC migration via a Cox- dependent mechanism. I will also determine if mice with null mutations in Cox1 and Cox2 have HSCR-like distal bowel aganglionosis. Finally, mice genetically predisposed to HSCR due to mutations in genes important for normal ENCC migration will be exposed to ibuprofen in utero during the critical period of ENS development. These experiments will examine if ibuprofen could be a common and avoidable exposure during pregnancy that increases HSCR risk by reducing the efficiency of ENCC migration.

描述(申请人提供)：出生缺陷是美国一岁以下儿童最常见的死亡原因。减少特定出生缺陷的发病率和死亡率的最好方法是制定预防它们发生的策略，叶酸补充导致的神经管缺陷的减少就是例证。先天性巨结肠(HSCR)是一种肠道神经系统的先天缺陷，当肠道神经峰源细胞(ENCC)不能一直迁移到肠道末端时就会发生这种疾病。由此导致的远端肠道无神经节细胞增多症导致功能性梗阻，导致呕吐、腹胀并容易过早死亡。有许多蛋白质对正常的ENCC迁移很重要，这些基因的突变会增加HSCR的风险，但这些突变都不会在所有受影响的儿童中引起疾病。因此，患有HSCR的儿童必须有进一步增加他们HSCR风险的额外因素。为了制定减少HSCR发生的策略，重要的是确定可变因素，例如母亲的饮食或药物，这些因素可能与基因缺陷结合在一起导致出生时健康的儿童患病。布洛芬是一种通常用于妊娠的非类固醇抗炎药(NSAID)，它在斑马鱼药物筛选中抑制ENCC迁移，导致受影响鱼类的HSCR样远端肠道无神经节细胞增多症。在哺乳动物原代培养中的实验证实，布洛芬减缓了ENCC的迁移，并似乎扰乱了有效迁移所需的肌动蛋白细胞骨架和焦点黏附调节。这项建议的目的是确定布洛芬抑制ENCC迁移的生化机制，并确定布洛芬是否增加了遗传易感性个体发生HSCR的可能性。布洛芬破坏抑制ENCC迁移的生化机制将使用尖端的荧光标记蛋白质的延时活细胞成像进行研究。这些研究将有助于深入分析ENCC肌动蛋白突起和局部粘连的动力学。此外，来自Cox1和Cox2零突变的转基因动物的ENCC将通过免疫组织化学和时间推移成像进行分析，以确定布洛芬是否通过COX依赖的机制抑制ENCC的迁移。我还将确定COX1和COX2零突变的小鼠是否患有HSCR样的远端肠道无神经节细胞增多症。最后，由于对正常ENCC迁移至关重要的基因突变而易患HSCR的小鼠，将在ENS发育的关键时期在子宫内暴露于布洛芬。这些实验将检查布洛芬是否可能是怀孕期间常见的和可避免的暴露，通过降低ENCC迁移的效率增加HSCR风险。