Identification oftargetproteins for endocrine disrupters in the testis

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具有核心重要性。携带温度敏感型猿猴病毒40大T抗原基因的转基因小鼠和大鼠可用于从难以体外培养的器官和组织中建立细胞系。转基因动物产生了许多条件永生化细胞系，如小鼠胃上皮细胞、睾丸支持细胞和间质细胞。DNA微阵列技术有着广泛的应用，它的力量是针对全球基因表达的研究。利用已建立的细胞系和DNA微阵列，我们鉴定了许多在细胞分化或细胞死亡过程中表达上调和下调的基因。我们利用双酚A(BPA)处理的小鼠Sertoli TTE3细胞的cDNA微阵列分析，详细检测了基因表达变化的时间进程。亚毒性剂量的双酚A(200μM)可引起细胞内钙离子瞬时升高，并呈时间依赖性地诱导78 kDa葡萄糖调节蛋白基因表达水平升高，提示双酚A可诱导内质网应激。在分析的865个基因中，有31个基因的表达水平有所上升。TaqMan分析证实CHOP、Fra-2、c-myc和鸟氨酸脱羧酶基因表达水平升高，表明CHOP-10是最敏感的基因。在稳定表达全长CHOP反义RNA的TTE3细胞中，CHOP蛋白的表达水平和BPA诱导的细胞损伤显著降低。结论：CHOP在双酚A诱导的支持细胞损伤中起关键作用。

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.