DEVELOPMENT OF ADRENAL CORTICAL IMMORTALIZED CELLS

我们建议从肾上腺带分离出细胞系。 保持重要表型的肾小球和束状 天然组织。我们将使用两种基本方法来生产这些产品 细胞系。首先，我们将癌基因导入纯化的分离株中 使用逆转录病毒载体的肾上腺皮质细胞。其次，我们将 产生在肾上腺特异表达癌基因的转基因小鼠 使用肾上腺特异性类固醇的启动子区域 21-羟基酶基因。几个癌基因将被利用，包括 温度敏感型突变体。将对生成的细胞系进行筛选 分化的肾上腺皮质细胞的最终产物的产生， 肾小球细胞的醛固酮和肾小球细胞的皮质酮 束状细胞。为了确定它们作为模型的适用性 分泌这些类固醇的天然肾上腺细胞系 进一步刻画的。他们对类固醇促分泌剂、受体的反应 曲目，激活细胞内第二信使(IP3，DAG， CA)，并将确定电生理特性。 将开发对这些细胞系进行遗传操作的方法 并应用于1)局部影响的调查 肾素-血管紧张素系统对醛固酮分泌的影响 刺激类固醇分泌的细胞内途径和3) 细胞对环境变化的长期适应机制 (作为肾上腺敏感度变化的模型 饮食中的钠或钾)。我们也希望这些细胞系能大大提高 促进目前对急性分散的研究进行的研究 肾上腺细胞。这些研究将进一步加深我们对 饮食控制引起的肾上腺敏感性的变化和 肾上腺在血压稳态中的作用。

We propose to produce cell lines derived from the adrenal zona glomerulosa and fasiculata which maintain important phenotypes of the natural tissue. We will use 2 basic methods for the production of these cell lines. First, we will introduce oncogenes into purified isolated adrenal cortical cells using a retroviral vector. Secondly, we will produce transgenic mice specifically expressing oncogenes in the adrenal cortex by using the promoter region from the adrenal specific steroid 21-hydroxylase gene. Several oncogenes will be utilized including temperature sensitive mutants. Resulting cell lines will be screened for production of end products of differentiated adrenal cortical cells, aldosterone for the glomerulosa cells and corticosterone for the fasiculata cells. In order to determine their suitability as models for the natural adrenal cells, cell lines secreting these steroids will be further characterized. Their responses to steroid secretagogue, receptor repertoire, activation of intracellular second messengers (IP3, DAG, Ca++) , and electrophysiologic properties will be determined. Methods for genetic manipulation of these cell lines will be developed and applied to 1) investigation of the effects of the local renin-angiotensin system on aldosterone secretion, 2) elucidation of the intracellular pathways leading to stimulation of steroid secretion and 3) the mechanism of long term cellular adaption to changes in environment (as a model for changes in adrenal sensitivity caused by alterations in dietary Na+ or K+). We also expect these cell lines to greatly facilitate studies which are currently performed on acutely dispersed adrenal cells. These studies will further our understanding of the changes in adrenal sensitivity caused by dietary manipulations and the role of the adrenal in blood pressure homeostasis.