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Analysis of cardiovascular development and pathophysiology by gene engineering of the endothelin system in mice

通过内皮素系统基因工程分析小鼠心血管发育和病理生理学

基本信息

批准号：
18390229
负责人：
KURIHARA Hiroki
金额：
$ 11.36万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

We have achieved the following results in regard to the molecular mechanisms underlying the involvement of the endothelin (ET) system in cardiovascular and craniofacial development and pathophysiology.1. We have established the Cre-recombinase-mediated gene knock-in system in mouse ES cells, in which we can systematically replace the ET-A receptor (STAR) gene with exogenous genes.2. By using this system, we knocked-in cDNAs encoding the ET-B receptor (ETBR) and ETAR-ETBR chimeric receptors. These experiments have revealed that i) both STAR subtype-selective and nonselective signaling are involved in the craniofacial development and ii) the induction of Dlx5/Dlx6 homeobox genes and subsequent specification of the mandibular identity is mediated by the STAR subtype-selective, Gq/G11-mediated signaling pathway.3. Knock-in of the lacZ gene revealed a possible novel cell lineage originating within the cardiac crescent and contributing to the early cardiovascular development.4. Knock-in of EGFP enabled us to visualize the STAR-positive cells in situ in mice, which is useful for the analysis of the dynamics of STAR-positive cells (e.g. smooth muscle cells) in embryonic vascular formation and in the physiological and pathophysiological processes.5. We have identified Calpain-6 as a target molecule of the ET-1/STAR signaling pathway in craniofacial development. We have discovered the novel function of Calpain-6 in the stabilization of microtubules and actin organization involved in cellular morphology and motility. The studies on the developmental role of Calpain-6 are starting with its knockout mice established recently.These achievements contribute to the understanding of the mechanisms of the cardiovascular and craniofacial development and pave a way to the development of experimental systems applicable to the studies on (patho)physiology involving the ET system.

我们在内皮素（ET）系统参与心血管和颅面发育和病理生理的分子机制方面取得了以下结果.建立了Cre重组酶介导的小鼠ES细胞基因敲入系统，系统地将ET-A受体（星星）基因替换为外源基因.通过使用该系统，我们敲入编码ET-B受体（ETBR）和ETAR-ETBR嵌合受体的cDNA。这些实验表明：i）星星亚型选择性和非选择性信号通路均参与颅面发育; ii）Dlx 5/Dlx 6同源框基因的诱导和随后的下颌骨身份的特化是由星星亚型选择性、Gq/G11介导的信号通路介导的. lacZ基因的敲入揭示了一个可能的新的细胞谱系起源于心脏新月体，并有助于早期心血管发育. EGFP的敲入使我们能够在小鼠体内原位观察STAR阳性细胞，这对于分析STAR阳性细胞（例如平滑肌细胞）在胚胎血管形成以及生理和病理生理过程中的动力学是有用的.我们已经确定Calpain-6是颅面发育中ET-1/星星信号通路的靶分子。我们已经发现了钙蛋白酶-6在稳定微管和肌动蛋白组织中的新功能，这些微管和肌动蛋白组织参与细胞形态和运动。近年来，Calpain-6基因敲除小鼠的建立为研究Calpain-6在发育中的作用奠定了基础，这些研究成果有助于深入了解心血管和颅面发育的机制，并为建立与ET系统相关的病理生理学研究的实验系统奠定了基础。