CONGENITAL MEGACOLON: TISSUE INTERACTIONS IN DEVELOPMENT

先天性巨结肠：发育过程中的组织相互作用

• 批准号: 3314763
• 负责人: VIRGINIA M TENNYSON
• 金额: $ 21.4万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-12-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3314763
• 关键词: binding proteins; cell adhesion; cell differentiation; cell migration; congenital megacolon; electron microscopy; extracellular matrix; immunocytochemistry; laboratory mouse; laboratory rat; laminin; molecular pathology; neural crest; tissue /cell culture

The enteric nervous system is formed by precursors that migrate to the bowel from the neural crest. In order to determine why crest-derived cells stop migrating and begin to differentiate in the gut, we have studied a mutant mouse, the lethal spotted (ls/ls), in which the terminal bowel is congenitally aganglionic. We have shown that the defect in these animals arises, not because their crest-derived cells are abnormal, but because neither these, nor any other source of crest-derived cells, is able to colonize the presumptive aganglionic ls/ls gut. Within this tissue, molecular components of basal laminae, including laminin, are overabundant and/or maldistributed. We have proposed that crest-derived cells, when they enter the bowel, acquire a nerve-related 110 kDa laminin receptor which, when activated, induces these cells to withdraw from the cell cycle, extend processes and cease migrating. The 110 kDa laminin binding site is not found on pre- or early migrating crest cells. We thus wish to test the hypothesis that the ls/ls defect is due to the abnormal extracellular matrix (ECM) of the presumptive aganglionic zone. We propose that migrating crest-derived cells stop, cease to proliferate, and extend processes prematurely when they encounter this ECM. As a result, the distal bowel does not become colonized by crest-derived precursors of neurons and glia, and there is no accumulation of neurons at the periphery of the abnormal zone. We will now test this hypothesis by using a purified population of crest-derived cells isolated from within the gut by magnetic immunoselection (employing antibodies to cell surface molecules expressed in the gut only by crest-derived cells). The effects of laminin and a variety of other defined substrates on the behavior of these cells in vitro will be ascertained. Parameters to be studied will include adhesion, migration, proliferation, neurite extension, and phenotypic expression. In addition to observation, quantitative measurements will be made of neural and glial expression, using immunocytochemical markers. Immunoselected enteric crest-derived cells will also be cultured on cyostat sections of control and presumptive aganglionic ls/ls bowel, in order to determine whether laminin or other molecules mimic the action of the abnormal ECM of the ls/ls gut on each the measured parameters of cell behavior. The effects on these parameters of synthetic peptides, which encompass neurite-promoting domains of laminin, antibodies to these domains, and antibodies to the 110 kDa protein will then be tested on immunoselected crest-derived cells cultured on laminin or cryostat sections of presumptive aganglionic ls/ls and control gut. Finally, the behavior of crest-derived cells and the effects of potential laminin antagonists will be analyzed by direct visualization using confocal fluorescence microscopy to observe the cells as they migrate in vitro within explants of fetal bowel from ls/ls and control mice.

肠神经系统是由前体细胞形成的， 从神经嵴上取下肠子 为了确定为什么冠状动脉 细胞停止迁移并开始在肠道中分化， 研究了一种突变小鼠，致命斑点（ls/ls），其中终端 肠道先天性无神经节 我们已经证明， 这些动物的出现，不是因为它们的冠状细胞异常， 但因为这些，也不是任何其他来源的嵴源细胞， 能够定殖于假定的无神经节的LS/LS肠道。 在这 基底层的组织、分子成分，包括层粘连蛋白， 过多和/或分布不均。 我们已经提出， 当细胞进入肠道时，获得神经相关的110 kDa层粘连蛋白 受体，当被激活时，诱导这些细胞从细胞中退出。 细胞周期，延长进程并停止迁移。 110 kDa层粘连蛋白 在迁移前或迁移早期的嵴细胞上未发现结合位点。 我们 因此希望检验LS/LS缺陷是由于 假定无神经节区的异常细胞外基质（ECM）。 我们认为迁移的嵴源性细胞停止增殖， 并在遇到此ECM时过早地扩展进程。 作为 结果，远端肠不会被冠状动脉源性 神经元和神经胶质的前体，并且没有神经元的积累 在异常区的外围。 我们现在要验证这个假设 通过使用分离自 通过磁性免疫选择（使用针对细胞的抗体） 肠中仅由嵴源性细胞表达的表面分子）。 的 层粘连蛋白和各种其他确定的底物对 将确定这些细胞在体外的行为。 的参数 研究将包括粘附、迁移、增殖、神经突 延伸和表型表达。 除了观察， 将对神经和神经胶质表达进行定量测量， 免疫细胞化学标记。 免疫选择性肠嵴衍生 细胞也将在对照和假定的细胞切片上培养。 无神经节LS/LS肠，以确定是否层粘连蛋白或其他 分子模拟ls/ls肠道的异常ECM对每个细胞的作用， 细胞行为的测量参数。 对这些参数的影响 的合成肽，其中包括神经突促进结构域的 层粘连蛋白，这些结构域的抗体，和110 kDa的抗体， 然后将在免疫选择的冠状细胞上测试蛋白质 在假定的无神经节的LS/LS的层粘连蛋白或低温恒温器切片上培养 控制肠道 最后，研究了脊源细胞的行为和 潜在的层粘连蛋白拮抗剂的作用将通过直接 使用共聚焦荧光显微镜观察细胞 因为它们在体外在来自LS/LS的胎儿肠的外植体内迁移， 对照小鼠。