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The Molecular Mechanism of Organ Regression during the Development

发育过程中器官退化的分子机制

基本信息

批准号：
14599007
负责人：
YAOITA Yoshio
金额：
$ 2.24万
依托单位：
Hiroshima University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

Many organs are formed in the process of the development, while unnecessary ones are eliminated. During the amphibian metamorphosis, tadpole tail and gills are regressing, as levels of plasma thyroid hormone are rising. By our research the following results are obtained.1.We injected dominant-negative thyroid hormone receptor gene together with a maker gene into tail muscle cells in living tadpoles to interrupt thyroid hormone signaling, thereby demonstrating that muscle cell death in tadpole tail commit suicide till NF-stage 62. Furthermore, we have shown that muscle cell death in rapidly shortening tails after NF-stage 62 is executed not only by suicide, but also by murder mechanism where cells lose the anchorage and die by the degradation of the extracellular matrix due to the increase of the matrix metalloproteinases.2.The matrix metalloproteinases(MMP) are believed to play a main role in the murder mechanism during the tail regression. A MMP inhibitor represses completely the resolution of the notochord that is constituted by the basement membrane, but partially the regression of tail. This result supports that dual mechanisms, suicide and murder, contribute to tail regression.3.We have isolated two clones by the subtraction library and differential hybridization as genes correlated with tail muscle cell death, obtained their full-length cDNAs, and analyzed. Both of them are actin-binding proteins, and not considered as suicide genes, because cell survival was not reduced by their overexpression in tadpole tail-derived myoblastic cell line.4.A cDNA library has been constructed by placing cDNAs downstream of the tetracycline-inducible promoter. Tadpoles are coinjected with clones of the cDNA library and a reporter gene, and reared in the presence of tetracycline derivative, doxycycline. We are searching for cDNA clones which diminishes the expression of reporter gene after doxycycline treatment.

许多器官是在发育过程中形成的，而不必要的器官则被消除了。在两栖动物的变态过程中，由于血浆甲状腺激素水平的上升，蝌蚪的尾巴和鳃正在退化。通过我们的研究，我们获得了以下结果：1.将显性-负性甲状腺激素受体基因和标记基因一起注射到活着的蝌蚪尾部肌肉细胞中，阻断甲状腺激素信号转导，从而证明蝌蚪尾部肌肉细胞死亡是自杀的，直到NF-62阶段。此外，我们还发现，在NF-Stage 62之后，快速缩短的尾巴中的肌细胞死亡不仅是自杀，而且是通过谋杀机制执行的，其中细胞失去锚定，由于基质金属蛋白酶的增加而导致细胞外基质的降解而死亡。2.在尾部退化过程中，基质金属蛋白酶(MMPs)被认为在谋杀机制中起主要作用。基质金属蛋白酶抑制剂完全抑制由基底膜构成的脊索的分解，但部分抑制尾部的退缩。这一结果支持自杀和谋杀两种机制在尾部回归中的作用。3.通过消减文库和差异杂交技术分离了两个与尾部肌肉细胞死亡相关的基因克隆，获得了它们的全长cDNA，并进行了分析。这两个基因都是肌动蛋白结合蛋白，不被认为是自杀基因，因为它们在蝌蚪尾部来源的成肌细胞系中的过度表达并没有降低细胞存活率。4.将cDNA放置在四环素诱导启动子的下游，构建了一个cDNA文库。用克隆的cDNA文库和一个报告基因共注射蝌蚪，并在四环素衍生物多西环素的存在下饲养。我们正在寻找在多西环素治疗后降低报告基因表达的cDNA克隆。