ROLE OF EXTRACELLULAR MATRIX REMODELING IN MANDIBULAR MORPHOGENESIS

细胞外基质重塑在下颌形态发生中的作用

• 批准号: 6270323
• 负责人: ZENA WERB
• 金额: $ 20.43万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-12-01 至 1999-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6270323
• 关键词: antibody; antisense nucleic acid; bone development; cartilage development; cell differentiation; complementary RNA; embryo /fetus tissue /cell culture; embryogenesis; extracellular matrix; gene expression; genetically modified animals; growth factor; histogenesis; immunofluorescence technique; in situ hybridization; laboratory mouse; mandible /maxilla; metalloenzyme; nucleic acid hybridization; organ culture; osteogenesis; phenotype; physiologic bone resorption; polymerase chain reaction; tissue /cell culture; tissue inhibitor of metalloproteinases; western blottings

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.

发育生物学的一个主要问题是时间和空间如何 指令在胚胎发生过程中被转化为形式。 第一个 鳃弓经历下颌结构的形态发生， 包括来自无定形间充质的下颌软骨和骨 体内从胚胎 (E)10 天到 14 天。 在E10组织中的程序 形态发生已确定，并在随后的培养 9 天中形成所有 下颌骨组织在无血清、化学成分确定的培养基中。 因此，该系统非常适合确定局部因素 调节骨和软骨的形态发生。 细胞外基质 重塑被认为在成长和发展中发挥着重要作用 结缔组织的发育。 该提案的目的是 确定细胞外基质重塑是否由 金属蛋白酶及其抑制剂，已知是主要的 骨和软骨的成分，在形成和形态中发挥作用 骨和软骨的细胞外基质成分 下颌骨发育。 为了检验这个假设，必须获得 金属蛋白酶表达的定量和定性数据 （MMP）和金属蛋白酶抑制剂（TIMP）在下颌骨 体内和培养中的发育。 该方法将起诉RT-PCR 蛋白酶和抑制剂 mRNA 转录物的初步鉴定， 然后通过酶促和分析这些基因的蛋白质产物 免疫学手段。 一旦这些 MMP 和 TIMP 被识别并 他们定义的表达的时间模式，选择的表达 MMP 和 TIMP 将被映射到软骨形成和/或成骨病灶 体内发育过程中的原位杂交和免疫细胞化学 和文化方面。 那么这些MMP和TIMP的功能就是 研究过。 一种方法将涉及添加纯化的或重组的 将 MMP 或 TIMP 加入下颌骨培养物中，看看这些是否会扰乱形态发生。 第二种方法将涉及产生亚形态表型 针对 MMP 或 TIMP 的阻断抗体或反义寡核苷酸来查看 如果这些影响形态发生。 第三种方法涉及调制 通过添加外源生长来影响下颌生长和形态发生 和分化因子或通过抗体消除这些因子 或反义寡核苷酸，然后确定其效果 关于 MMP 和 TIMP 表达，以及关于 MMP 和 TIMP 表达的时间、位置和表达 软骨形成和骨形成。 这些实验应该会导致 了解细胞外参与的分子因素 一般而言，骨和软骨形态发生的基质方面 尤其是下颌骨，并导致新的治疗策略 下颌畸形以及骨和软骨修复。