Studies on functional xonation of adrenocortical cells

肾上腺皮质细胞功能性氧化的研究

基本信息

批准号：
09044333
负责人：
ISIMURA Yuzuru
金额：
$ 1.15万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1997
资助国家：
日本
起止时间：
1997 至无数据
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09044333/
关键词：
adrenal cortex mineralocorticoid glucocorticoid thymidine kinase transgenic mice SV40 large T antigen Y-1 cells AP-1 transcription factor 副賢皮質

项目摘要

Mechanisms for the functional zonation of adrenocortical cells were studied using mice that were genetically manipulated. Findings obtained from research results are described below.1. Production of mice whose mineralocorticoid synthesizing cell-zone in the adrenal cortex are conditionally ablated. Thymidine kinase gene of herpes simplex virus was inserted into the locus at mineralocorticoid synthase gene of embryonic stem cells by transfection with a targeting vector. Recombinant embryonic stem cell clones were successfully obtained, and introduction of them into mouse blastocysts for chimeric animals are undergoing.2. Establishment of adrenocortical precursor cell-lines. Adrenocortical cells from transgenic mice which harbor an immortalizing gene encoding temperature sensitive SV40 large T antigen, were cultured and cloned with a conventional methods. The cell lines were characterized for expression of various steroidogenic genes by analyzing their mRNAs. The results indicated that a cell-line was undifferentiated at the permissive temperature and express glucocorticoid synthase gene at the nonpermissive temperature. Thus, and adrenocortical precursor cell-line which was able to differentiate upon a temperature shift was established.3. Molecular mechanisms by which adrenocorticotropic hormone (ACTH) stimulates glucocorticoid synthase gene expression. Using mouse adrenocortical Y-1 cells, ACTH was found to regulate transcription of the gene by causing compositional changes in AP-1 transcripotion factors in the cells via a cAMP-dependent pathway.4. Histochemical analyzes of mouse adrenal cortex. Immunohistochemical and in situ hybridization analyzes of mouse adrenal cortex showed the presence of an undifferentiated cell layr which express neither mineralonor glucocorticoid synthase.

使用基因操纵的小鼠研究了肾上腺皮质细胞功能分区的机制。研究结果如下： 1．生产肾上腺皮质中盐皮质激素合成细胞区被有条件消除的小鼠。通过靶向载体转染，将单纯疱疹病毒的胸苷激酶基因插入胚胎干细胞的盐皮质激素合酶基因位点。成功获得重组胚胎干细胞克隆，并正在将其导入小鼠囊胚中构建嵌合动物。 2．肾上腺皮质前体细胞系的建立。用常规方法培养并克隆来自转基因小鼠的肾上腺皮质细胞，所述转基因小鼠含有编码温度敏感的SV40大T抗原的永生化基因。通过分析细胞系的 mRNA，对细胞系的各种类固醇生成基因的表达进行表征。结果表明，细胞系在允许温度下未分化，而在非允许温度下表达糖皮质激素合酶基因。由此，建立了能够随温度变化而分化的肾上腺皮质前体细胞系。 3．促肾上腺皮质激素（ACTH）刺激糖皮质激素合酶基因表达的分子机制。使用小鼠肾上腺皮质Y-1细胞，发现ACTH通过cAMP依赖性途径引起细胞内AP-1转录因子的组成变化来调节基因转录。4．小鼠肾上腺皮质的组织化学分析。小鼠肾上腺皮质的免疫组织化学和原位杂交分析显示存在未分化的细胞层，其既不表达盐也不表达糖皮质激素合酶。