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StAR gene structure-function analysis using immortalized adrenal steroidogenic cell line

使用永生化肾上腺类固醇生成细胞系进行 StAR 基因结构功能分析

基本信息

批准号：
17591113
负责人：
HASEGAWA Tomonobu
金额：
$ 2.24万
依托单位：
KEIO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

The aim of this study was to elucidate the structure-function relationship of mouse steroidogenic acute regulatory protein (StAR) gene in steroidogenic cell line in vitro.1.We established two types of immortalized adrenal steroidogenic cell lines ; one from wild type mice (wild cell line) and the other from StAR knockout mice (StAR (-) cell line), using temperature sensitive SV40 large T antigen transgenic mice.2.We transfected wild type StAR gene into StAR (-) cell line by calcium phosphate method. We then confirmed the expression of wild StAR mRNA by RT-PCR.3.We generated five mutated StAR genes by mutagenesis ; (1)one without mitochondrial targeting sequence, (2)one without last 28 amino acid, which is the identical mutant most commonly found in human disease due to StAR gene mutation, (3)one with S57A (putative phosphorylation site was substituted from serine to alamine), (4)one with S196A (another putative phosphorylation site was substituted from serine to alamine), and (5)one with both S57A and S196A. Each mutated StAR gene was introduced into expression vector.4.We are transfecting these mutant StAR gene into Star (-) cell line by calcium phosphate method.5.After confirming the expression of each mutated StAR gene, we will measure the adrenal steroid hormone in conditioned media from StAR (-) cell line, into which either wild type or each mutant StAR gene was transfected.These studies will highlight the pivotal functional sites in StAR gene structure.

本研究的目的是在体外阐明小鼠类固醇生成急性调节蛋白(StAR)基因在类固醇生成细胞系中的结构与功能关系。1．建立了两类永生化肾上腺类固醇生成细胞系；2．一只来自野生型小鼠（野生细胞系），另一只来自StAR敲除小鼠（StAR（-）细胞系），使用温度敏感的SV40大T抗原转基因小鼠。2.我们通过磷酸钙法将野生型StAR基因转染到StAR（-）细胞系中。然后通过RT-PCR证实了野生StAR mRNA的表达。3.通过诱变产生了5个突变的StAR基因； (1) 一个没有线粒体靶向序列，(2) 一个没有最后 28 个氨基酸，这是由于 StAR 基因突变而在人类疾病中最常见的相同突变体，(3) 一个具有 S57A（假定的磷酸化位点从丝氨酸替换为丙胺），(4) 一个具有 S196A（另一个假定的磷酸化位点从丝氨酸替换为丙胺），以及 (5) 一个具有 S57A 和 S196A。将每个突变的StAR基因导入表达载体。4.我们通过磷酸钙法将这些突变的StAR基因转染到Star（-）细胞系中。5.在确认每个突变的StAR基因的表达后，我们将测量转染野生型或每个突变的StAR基因的StAR（-）细胞系的条件培养基中的肾上腺类固醇激素。这些研究将突出StAR基因中的关键功能位点结构。