CONGENITAL MEGACOLON: TISSUE INTERACTIONS IN DEVELOPMENT

肠道神经系统是由迁移到 从神经峰排出的肠道。为了确定为什么CREST派生出 细胞停止迁移，并开始在肠道中分化，我们有 研究了一只突变小鼠，致命斑点(ls/ls)，其中末端 肠道先天无神经节细胞。我们已经证明了 这些动物之所以出现，并不是因为它们的冠状突起细胞不正常， 但因为无论是这些，还是任何其他来源的冠状突起细胞， 能够在推定的无神经节细胞的肠道上定居。在这个范围内 组织中，基底层的分子成分，包括层粘连蛋白，是 数量过多和/或分布不均。我们已经提出了由CREST派生的 当细胞进入肠道时，它们会获得一种与神经相关的110 kDa层粘连蛋白 受体，当被激活时，诱导这些细胞从 细胞周期，延长过程和停止迁移。110 kDa的层压板 在迁移前或早期迁移的脊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 推测为无神经节带的异常细胞外基质。 我们建议迁移的CREST来源的细胞停止，停止增殖， 并在遇到这种ECM时过早地扩展流程。作为一名 结果，远端肠道不会被CREST来源的 神经元和胶质细胞的前体，没有神经元的积聚 在异常带的外围。我们现在将检验这一假设 通过使用纯化的CREST来源的细胞群从 在肠道内通过磁性免疫选择(使用细胞抗体 表面分子仅在肠道中表达，由冠状突起细胞表达)。这个 层粘连蛋白和其他各种已定义的底物对 这些细胞在体外的行为将被确定。参数应为 研究内容包括黏附、迁移、增殖、轴突 外延和表型表达。除了观察之外， 将对神经和神经胶质的表达进行定量测量， 使用免疫细胞化学标记物。免疫选择肠冠源性 细胞也将在对照和假定的Cyostat切片上培养 无神经节细胞肠管，以确定层粘连蛋白或其他 分子模拟ls/ls肠道异常ECM对每个 测量的细胞行为参数。对这些参数的影响 合成的多肽，它包含神经突起促进结构域 层粘连蛋白、针对这些结构域的抗体以及针对110 kDa的抗体 蛋白质随后将在免疫选择的CREST来源的细胞上进行测试 在假定无神经节细胞的层粘连蛋白或冷冻切片上培养 和控制本能。最后，CREST派生的细胞的行为和 潜在的层粘连蛋白拮抗剂的作用将通过直接分析 用共聚焦荧光显微镜观察细胞的可视化 当它们在体外迁移到来自ls/ls和 控制小鼠。

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.