ROLE OF EXTRACELLULAR MATRIX REMODELING IN MANDIBULAR MORPHOGENESIS

发育生物学中的一个主要问题是时间和空间如何 指令在胚胎发育过程中被翻译成形式。第一 颧弓对下颌骨的结构进行形态发生， 包括来自无定形间充质的下颌软骨和骨 在体内从胚胎第10天到第14天。在E10组织中的程序 形态发生被设定，在随后9天的培养中，所有的形态 在无血清、化学定义的培养液中的下颌骨组织。 因此，该系统非常适合于确定 调节骨和软骨的形态发生。细胞外基质 重塑被认为在生长和发展中起着重要作用 结缔组织的发育。这项建议的目的是 确定细胞外基质重塑是否由 金属蛋白酶及其抑制物，已知是主要的 骨和软骨的成分，在形成和形成中发挥作用 骨和软骨的细胞外基质成分 下颌骨发育。要检验这一假设，必须获得 金属蛋白酶表达的定量和定性数据 (MMPs和TIMPs)在下颌骨中的表达 在体内和在培养中发育。该方法将起诉RT-PCR 蛋白水解酶和抑制物基因转录本的初步鉴定， 然后对这些基因的蛋白产物进行了酶和蛋白质分析 免疫学手段。一旦确定了这些MMP和TIMP， 他们对表达的时间模式进行了界定，对表达的选择 MMPs和TIMP将通过以下方式映射到软骨和/或成骨病灶 体内发育过程中的原位杂交和免疫细胞化学 在文化上也是。那么这些MMP和TIMP的功能将是 学习。一种方法将包括添加纯化的或重组的 MMPs或TIMP到下颌骨培养，看看这些是否扰乱形态发生。 第二种方法将包括产生一个亚形表型， 阻断针对MMPs或TIMPs的抗体或反义寡核苷酸 如果这些影响了形态发生。第三种方法将涉及调制 外源生长对下颌骨生长和形态发生的影响 和分化因子，或通过抗体去除这些因子 或反义寡核苷酸，然后测定它们的作用 MMPs和TIMP的表达及其时间、位置和表达的关系 软骨生成和成骨。这些实验应该会导致一个 对细胞外参与的分子因素的认识 骨和软骨形态发生的基质方面，一般和 尤其是下颌骨，并导致了新的治疗策略 下颌畸形及骨和软骨修复。

A major issue in developmental biology is how the temporal and spatial instructions are translated to form during embryogenesis. The first branchial arch undergoes morphogenesis to structures of the lower jaw, including the mandibular cartilage and bone from an amorphous mesenchyme from embryonic day (E)10 to 14 in vivo. In E10 tissue the program of morphogenesis is set, and in subsequent culture for 9 days forms all the tissues of the mandible in serum-less, chemically defined culture medium. Thus, this system is ideal for determining the local factors that regulate the morphogenesis of bone and cartilage. Extracellular matrix remodeling is believed to play a significant role in growth and development of connective tissues. The aim of this proposal is to determine whether extracellular matrix remodeling mediated by metalloproteinases and their inhibitors, which are known to be major components of bone and cartilage, plays a role in the formation and form of the extracellular matrix constituents of bone and cartilage during mandible development. To test this hypothesis it is essential to obtain quantitative and qualitative data on the expression of metalloproteinases (MMPs) and metalloproteinase inhibitors (TIMPs) during mandibular development in vivo and in culture. The approach will sue RT-PCR for initial identification of proteinase and inhibitor mRNA transcripts, followed by analysis of protein products of these genes by enzymatic and immunological means. Once these MMPs and TIMPs have been identified and their temporal patterns of expression defined, the expression of selected MMPs and TIMPs will be mapped to chondrogenic and/or osteogenic foci by in situ hybridization and immunocytochemistry during development in vivo and in culture. Then the function of these MMPs and TIMPs will be studied. One approach will involve addition of purified or recombinant MMPs or TIMPs to mandible cultures to see if these perturb morphogenesis. A second approach will involve production of a hypomorphic phenotype with blocking antibodies or antisense oligonucleotides to MMPs or TIMPs to see if these affect morphogenesis. A third approach will involve modulation of mandibular growth and morphogenesis by addition of exogenous growth and differentiation factors or by ablation of these factors by antibodies or antisense oligonucleotides followed by determination of their effects on MMP and TIMP expression, and on timing, position, and expression of chondrogenesis and osteogenesis. These experiments should lead to an understanding of the molecular factors involved in the extracellular matrix aspects of bone and cartilage morphogenesis, in general, and in the mandible, in particular, and lead to novel therapeutic strategies for mandibular malformations and bone and cartilage repair.