Identification oftargetproteins for endocrine disrupters in the testis

睾丸内分泌干扰物靶蛋白的鉴定

• 批准号: 14572107
• 负责人: TABUCHI Yoshiaki
• 金额: $ 2.11万
• 依托单位: TOYAMA MEDICAL AND PHARMACEUTICAL UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14572107/
• 关键词: transgenic animals; temperature-sensitive oncogene; simian virus 40 large T-antigen; endocrine disruptor; bisphenol A; cDNA microarray; chop; gene expression; 精巣セルトリ細胞; cDNAマイクロアレイ; 小胞体ストレス; chop; GPR78; セルトリ細胞; DNAマイクロアレイ

The use of in vitro cell culture models has been of central importance in the development of cellular and molecular biology of Organs and tissues. Transgenic mouse and rat harboring temperature-sensitive simian virus 40 large T-antigen gene are useful for establishing cell lines from organs and tissues that have proved difficult to culture in vitro. Many conditionally immortalized cell lines such as mouse gastric epithelial, testicular Sertoli and Leydig cells were generated from the transgenic animals. DNA microarray technology has broad applications, and its power is directed toward the study of global gene expression. Using established cell lines and DNA microarrays, we identified many genes that were up-and down-regulated in the process of the cell differentiation or cell death.We examined the time course of changes in gene expression in detail using cDNA microarray analysis of mouse Sertoli TTE3 cells treated with bisphenol A(BPA). A subtoxic dose of BPA(200μM) transiently increased intracellular Ca^<2+> and time-dependently induced an increase in mRNA level of 78-kDa glucose-regulated protein, indicating that BPA induces endoplasmic reticulum stress. Of the 865 genes analyzed, 31 genes showed increased levels of expression. TaqMan analysis confirmed that the mRNA levels of chop, fra-2, c-myc, and ornithine decarboxylase were increased, and showed that chop-10 is the most sensitive gene. The expression level of chop protein and cell injury induced by BPA were significantly reduced in stable TTE3 cells overexpressing full-length chop antisense RNA. We conclude that chop plays a key role in Sertoli cell injury induced by BPA.

体外细胞培养模型的使用在器官和组织的细胞和分子生物学的发展中至关重要。转基因小鼠和大鼠含有温度敏感的猿猴病毒40大抗原基因可用于建立来自已证明在体外难以培养的器官和组织的细胞系。许多有条件永生的细胞系，例如小鼠胃上皮，验证的Sertoli和Leydig细胞是由转基因动物产生的。 DNA微阵列技术具有广泛的应用，其功率针对全球基因表达的研究。使用已建立的细胞系和DNA微阵列，我们确定了许多在细胞分化或细胞死亡过程中向上和下调的基因。我们研究了使用Bisphenol A（BPA）对小鼠Sertoli TTE3细胞的CDNA微阵列分析详细详细介绍基因表达的时间过程。 BPA（200μm）的潜毒剂量延伸性增加了细胞内Ca^<2+>和时间依赖性地诱导的78 kDa葡萄糖调节蛋白的mRNA水平升高，表明BPA诱导内质网应激。在分析的865个基因中，有31个基因显示出表达水平增加。 Taqman分析证实，CHOP，FRA-2，C-MYC和鸟氨酸脱羧酶的mRNA水平增加了，并表明Chop-10是最敏感的基因。在稳定的TTE3细胞中，BPA诱导的CHOP蛋白和细胞损伤的表达水平显着降低，过表达全长CHOP反义RNA。我们包括CHOP在BPA引起的Sertoli细胞损伤中起关键作用。

项目成果

Tabuchi et al.(3 authors): "DNA microarray analysis of differentially expressed genes responsive to bisphenol A an alkylphenol derivative, in an in vitro mouse Sertoli cell model"Jpn.J.Pharmacol.. 89. 413-416 (2002)

Tabuchi等人（3位作者）：“在体外小鼠支持细胞模型中对双酚A（一种烷基酚衍生物）反应的差异表达基因的DNA微阵列分析”Jpn.J.Pharmacol.. 89. 413-416 (2002)

Tabuchi Y.et al.(9 authors): "New gastric epithelial cell lines from mice transgenic for temperature-sensitive simian virus 40 large Tantigen gene exhibit distinct types of cell differentiation"Digestion. 67. 71-81 (2003)

Tabuchi Y.等人（9位作者）：“来自温度敏感猿病毒40大Tantigen基因转基因小鼠的新胃上皮细胞系表现出不同类型的细胞分化”消化。

Tabuchi et al.(7 authors): "Development of the conditionally immortalized testicular Sertoli cell line TTE3 expressing sertoli cell-specific genes from mice transgenic for temperature-sensitive simian virus 40 large T-antigen gene"J.Urol.. 167. 1538-1545

Tabuchi 等人（7 位作者）：“从温度敏感猿病毒 40 大 T 抗原基因转基因小鼠中开发表达支持细胞特异性基因的条件永生化睾丸支持细胞系 TTE3”J.Urol.. 167. 1538

Tabuchi, Y., Kondo, T.: "cDNA microarray analysis reveals chop-10 plays a key role in Sertoli cell death induced by bisphenol A"Biochem.Biophys.Res.Commun.. 305. 54-61 (2003)

Tabuchi, Y., Kondo, T.：“cDNA 微阵列分析表明，chop-10 在双酚 A 诱导的支持细胞死亡中发挥着关键作用”Biochem.Biophys.Res.Commun.. 305. 54-61 (2003)

Tabuchi, Y.et al.(3 authors): "DNA microarray analysis of differentially expressed genes responsive to bisphenol A, an alkylphenol derivative, in an in vitro mouse Sertoli cell model"Jpn. J. Pharmacol.. 89. 413-416 (2002)

Tabuchi, Y. 等人（3 位作者）：“在体外小鼠支持细胞模型中对双酚 A（一种烷基酚衍生物）响应的差异表达基因的 DNA 微阵列分析”Jpn。