Studies on functional xonation of adrenocortical cells

肾上腺皮质细胞的功能分区的机制进行了研究，使用基因操作的小鼠。从研究结果中得出的结论如下。肾上腺皮质中盐皮质激素合成细胞带被条件性消融的小鼠的产生。用靶向载体将单纯疱疹病毒胸苷激酶基因插入胚胎干细胞盐皮质激素合成酶基因位点。成功获得了重组胚胎干细胞克隆，并将其导入小鼠囊胚中用于嵌合动物.肾上腺皮质前体细胞系的建立。用常规方法培养和克隆了来自转基因小鼠的肾上腺皮质细胞，所述转基因小鼠携带编码温度敏感性SV 40大T抗原的永生化基因。通过分析它们的mRNA来表征细胞系的各种类固醇生成基因的表达。结果表明，该细胞系在允许温度下未分化，在非允许温度下表达糖皮质激素合成酶基因。从而建立了一种能够在温度变化下分化的肾上腺皮质前体细胞系.促肾上腺皮质激素（ACTH）刺激糖皮质激素合成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中AP-1转录因子的组成变化来调节基因的转录.小鼠肾上腺皮质的组织化学分析。免疫组织化学和原位杂交分析表明，小鼠肾上腺皮质存在一个未分化的细胞层，既不表达mineralon，也不表达糖皮质激素合成酶。

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.