TRANSGENIC MURINE MODELS TO STUDY HIRSCHSPRUNGS DISEASE

研究先天性巨结肠症的转基因小鼠模型

• 批准号: 2145457
• 负责人: RAJ P. KAPUR
• 金额: $ 13.3万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2145457
• 关键词: autoradiography; cell differentiation; cell migration; cellular pathology; congenital megacolon; cytogenetics; electron microscopy; gene mutation; genetically modified animals; immunocytochemistry; laboratory mouse; light microscopy; model design /development; molecular pathology; neurons; pathologic process

Hirschsprung's disease (HD), congenital absence of neurons in the distal intestinal tract, is a potentially lethal birth defect that afflicts 1/5000 liveborn humans. HD is thought to be a consequence of failed colonization of the hindgut by ganglion cell precursors (neuroblasts) derived from the neural crest. The processes which regulate the migration, proliferation, and differentiation of enteric neuroblasts are not understood, but probably involve dynamic interactions between these cells and the surrounding intestinal mesenchyme. Lethal spotted (ls), piebald lethal (sl), and dominant megacolon (Dom) are three alleles associated with HD in mice. I hope to learn more about the pathogenesis of HD through study of these murine models. In the proposed studies, enteric neuroblasts, their microenvironment, and interactions between the two will be examined by using expression of the transgene, D/beta/H- nlacZ, as a marker of neuroblasts in wild-type mice and ls/ls, sl/sl, and Dom/+ embryos. Use of the transgene is a novel approach which permits positive identification of "undifferentiated" enteric neuroblasts in intact gut and histological sections during the critical stages of embryogenesis when the gut is colonized. A combination of immunohistochemical, ultrastructural, autoradiographic techniques will be applied to define morphological and biochemical features which distinguish enteric neuroblasts and to identify possible differences in these cells or surrounding mesenchyme between wild-type, ls/ls, sl/sl, and Dom/+ embryos. The nature of the defect in sl/sl and Dom/+ embryos will be examined by examining chimeric mice produced by aggregating transgenic mutant embryos with non-transgenic wild-type embryos. We recently published such studies of ls/ls-wild type chimeras which demonstrated that the primary defect caused by the ls mutation is not cell autonomous (intrinsic to migrating neuroblasts), but is environmental (extrinsic anomalies in the milieu through which neuroblasts migrate). The effects of environmental manipulations on neuroblast colonization in wild-type and mutant embryonic intestines engrafted under the renal capsule will also be investigated with the intent of finding growth conditions that "rescue" the mutant phenotype. The cumulative results of these studies will provide insight into the pathogenesis of HD and direct future studies to identify specific genetic or environmental factors responsible for HD in humans.

先天性巨结肠（HD），远端神经元先天性缺失 肠道，是一种潜在的致命的出生缺陷， 1/5000活产人类。 HD被认为是失败的后果 神经节细胞前体（成神经细胞）在后肠的定植 源自神经嵴 调节生物多样性的过程 肠神经母细胞的迁移、增殖和分化是 不理解，但可能涉及这些之间的动态相互作用， 细胞和周围的肠间充质。 致命斑点（LS）， 花斑致死型（sl）和显性巨结肠型（Dom）为3个等位基因 与小鼠HD相关。 希望能进一步了解发病机理 通过对这些小鼠模型的研究， 在拟议的研究中， 肠神经母细胞，它们的微环境，以及 两个将通过使用转基因D/β/H-的表达来检查。 nlacZ作为野生型小鼠和ls/ls、sl/sl中神经母细胞的标志物， Dom/+胚胎。 使用转基因是一种新的方法， 阳性鉴定“未分化”肠神经母细胞， 完整的肠道和组织切片在关键阶段， 当肠道被殖民时，胚胎发生。 的组合 免疫组织化学、超微结构、放射自显影技术将 应用于定义形态学和生物化学特征， 区分肠神经母细胞，并确定可能的差异， 这些细胞或周围间充质在野生型，LS/LS，SL/SL， Dom/+胚胎。 sl/sl和Dom/+胚胎中缺陷的性质 将通过检查由聚集产生的嵌合小鼠来检查 转基因突变体胚胎与非转基因野生型胚胎。 我们 最近发表了这样的LS/LS-野生型嵌合体的研究， 证明了由ls突变引起的主要缺陷不是 细胞自主（固有迁移神经母细胞），但 环境（环境中的外在异常， 成神经细胞迁移）。 环境操纵对 神经母细胞在野生型和突变型胚胎肠中的定殖 移植到肾包膜下的移植物也将用 目的是寻找“拯救”突变表型的生长条件。 这些研究的累积结果将提供深入了解 HD的发病机制，并指导未来的研究，以确定特定的遗传 或环境因素导致人类HD。