NUTRITIONAL STUDIES OF TOOTH DEVELOPMENT IN VITRO

牙齿的发育是一个复杂的细胞分裂过程， 分化、形态发生和组织特异性细胞外基质 生物矿化本研究项目的主要目标是 确定内源性调节因子如何作为自分泌和/或 旁分泌介质来控制组织特异性釉质生物矿化。 基于初步证据表明胰岛素和胰岛素- 类生长因子（IGF-II）显着增加釉质 生物矿化，我们建议测试的假设，胰岛素和/或 IGF配体及其同源受体通过信号转导 过程控制釉质生物矿化的速率和量。一 不含外源性血清或血浆简单体外器官培养系统 补充剂提供了一个对照模型来测试我们的假设。的 以下三个主要的具体目标旨在检验我们的假设： (i)以确定表达和组织定位的时间， 胰岛素、胰岛素样生长因子（IGFI-I和II）及其同源物 受体（例如，胰岛素受体、IGF-I和IGF-II受体以及IGF 结合蛋白1和2）从帽到冠阶段的牙齿 （ii）确定胰岛素的控制水平，以及 IGF诱导釉质生物矿化（例如转录， 转录，翻译，翻译后）;和（iii）测试 假设胰岛素和/或IGF配体和/或 它们的同源受体阻碍釉质生物矿化。方法 所采用的方法包括显微解剖胚胎小鼠臼齿器官， 使用无血清和化学成分确定的培养基的器官培养 使用RT-PCR、定量PCR、反义翻译阻滞进行表型分型 检测、原位杂交、免疫细胞化学、光和电子 显微镜，电子衍射分析，和计算机辅助的三- 三维重建这项研究计划的长期目标是 是了解控制牙齿的调节机制 形态发生和釉质生物矿化，并应用这些知识 设计合理的策略来诊断和治疗牙齿组织， 疾病这些研究使用胚胎，胎儿和新生儿瑞士韦伯斯特 应变小鼠磨牙器官。

Tooth development is a complex schedule of cell division, differentiation, morphogenesis and tissue-specific extracellular matrix biomineralization. The major objective of this research project is to determine how endogenous regulatory factors act as autocrine and/or paracrine mediators to control tissue-specific enamel biomineralization. Based upon preliminary evidence demonstrating that insulin and insulin- like growth factor (IGF-II) significantly increase enamel biomineralization, we propose to test the hypothesis that insulin and/or IGF ligands and their cognate receptors through signal transduction processes control the rates and amount of enamel biomineralization. A simple in vitro organ culture system devoid of exogeneous serum or plasma supplementation provides a controlled model to test our hypothesis. The following three major Specific Aims are designed to test our hypothesis: (i) to identify the timing of expression and tissue localization for insulin, insulin-like growth factors (IGFI-I and II) and their cognate receptors (e.g., insulin receptor, IGF-I and IGF-II receptors, and IGF binding proteins 1 and 2) from cap through crown stages of tooth development; (ii) to determine the level(s) of control for insulin and IGF induction of enamel biomineralization (e.g. transcription, post- transcriptional, translation, post-translational); and (iii) to test the hypothesis that under-expression of insulin and/or IGF ligands and/or their cognate receptors retards enamel biomineralization. Methods employed include microdissection of embryonic mouse molar tooth organs, organ culture using serumless and chemically-defined medium, mRNA phenotyping using RT-PCR, quantitative PCR, antisense translation arrest assays, in situ hybridization, immunocytochemistry, light and electron microscopy, electron diffraction assays, and computer-assisted three- dimensional reconstructions. The long-term goals of this research program are to understand the regulatory mechanisms controlling tooth morphogenesis and enamel biomineralization, and to apply this knowledge to the design of rational strategies to diagnose and treat dental tissue diseases. These studies use embryonic, fetal and neonatal Swiss Webster strain mouse molar tooth organs.