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Elucidation of Cellular and Molecular Pathogenesis of Maxillo-Mandibular and Facial Malformation and Disorders : Use of Gene-Modified Mice

阐明上下颌和面部畸形和疾病的细胞和分子发病机制：使用基因修饰小鼠

基本信息

批准号：
13307052
负责人：
AOBA Takaaki
金额：
$ 33.95万
依托单位：
The Nippon Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2004
项目状态：
已结题

项目摘要

Our long-term strategy is to elucidate pathogenesis of varieties of malformations taking place in the maxillo-facial region. To this objective, the present project aimed to inspect phenotypic expression (and alterations) and relevant genetic network operating temporo-spatially during mouse embryonic development. We specifically focused attention on: (1) palategenesis and cleft formation, which is most frequently encountered among various malformations in humans, (2) development and anomalies of Meckel's cartilage and mandible, (3) early development and hypoplasia of temporomandibular joint (TMJ), and (4) cellular events involved in tongue development and its hypoplasia in Endothelin1 (-I-) mice. Cultures using the Trowell organ culture system were conducted to monitor in vitro cellular events in palatogenesis and tongue morphogenesis. Specimens in culture were maintained in a humidified incubator at 37℃ under 5% C0_2 using DMEM/Fl2 (1:1) medium. In 2004, gene expression patterns in secondary palate and tongue in vivo and in vitro were surveyed using 80 probes for RT-PCR and real-time PCR. We also continued analysis of gene expression in specific cell types, namely, snail and Rho family in medial edge epithelial cells during palategenesis and myogenic mater genes (Pax 3, MyoD, myogenin) in tongue primordium, which were isolated by laser-capture microdissection. Transgenic mice used were homozygous (-I-) for TGF-β, δEF1 or endothelin1, as well as Klotho (-I-) used for investigation of TMJ aging. Phenotypic features of these transgenic mice were scrutinized by means of histology and x-ray μCT in combination of 3D-reconstruction techniques. Imaging data collected during the funded period have been accumulated and tabulated in our database with the aid of a computer server, which was purchased in the last year. We at present are in the process to display them on the Web-site (http://www.ndu.ac.jp/~pathhome).

我们的长期策略是阐明发生在颌面部的各种畸形的发病机制。为此，本项目旨在研究小鼠胚胎发育过程中的表型表达（和改变）以及相关的时空遗传网络。我们特别关注：（1）腭发育和裂缝形成，这是最常见的各种畸形中的人类，（2）Meckel软骨和下颌骨的发育和异常，（3）颞下颌关节（TMJ）的早期发育和发育不良，（4）细胞事件参与舌发育和发育不良的内皮素1（-I-）小鼠。使用Trowell器官培养系统进行培养以监测腭发生和舌形态发生中的体外细胞事件。将培养物中的标本保存在37℃、5%CO_2的加湿培养箱中，使用DMEM/F12（1：1）培养基。在2004年，使用80个RT-PCR和实时荧光PCR探针，在体内和体外调查了次腭和舌的基因表达模式。我们还继续分析了特定细胞类型中的基因表达，即腭发育过程中内侧缘上皮细胞中的snail和Rho家族和舌原基中的肌原蛋白基因（Pax 3，MyoD，myogenin），这些基因是通过激光捕获显微切割分离的。所用的转基因小鼠是TGF-β、δ EF 1或内皮素1的纯合子（-I-），以及用于TMJ老化研究的Klotho（-I-）。通过组织学和x射线μCT结合3D重建技术对这些转基因小鼠的表型特征进行了详细检查。在去年购买的计算机服务器的帮助下，在资助期间收集的成像数据已经积累并在我们的数据库中制成表格。我们目前正在把这些资料放在网站上（http：//www.ndu.ac.jp/pathhome）。