Environmental biosensor of transgenic zebrafish

描述(申请人提供)：内分泌干扰物(EDCs)是一种可以模仿或改变体内荷尔蒙功能的物质。以前的研究大多使用细胞培养来检测内分泌细胞对特定激素反应的影响。我们建议使用转基因斑马鱼直接检测活体动物内分泌器官和激素反应的细微变化，以分析暴露于内分泌细胞后发生的细胞行为和基因表达变化。这些转基因斑马鱼分别在胰岛素启动子引导的胰腺、POMC启动子引导的脑垂体和卵黄蛋白B1启动子作用下对雌激素有反应的所有细胞中表达绿色荧光蛋白报告基因。将使用流式细胞术和微阵列和微SAGE进行基因表达分析，以在细胞水平上提供内分泌细胞激活的基因的概况。在分离的GFP表达细胞中，细胞因子、趋化因子、信号转导信使、蛋白酶、癌基因或细胞周期相关产物将成为研究的重点，并在自然雌激素暴露组和异源雌激素暴露组之间进行比较。为了开发更多的工具来研究内分泌器官发育和EDC诱导的反应，我们还将使用每个品系的转基因胚胎进行大规模的化合物筛选。在内分泌细胞谱系发育或雌激素反应中具有特定作用的小分子将被选为未来的研究对象。由于这项建议中将研究的生物过程高度保守，我们相信斑马鱼的研究结果最终应有助于我们了解环境压力如何影响人类内分泌细胞的发育和反应。

DESCRIPTION (provided by applicant): Endocrine disrupting chemicals (EDCs) are substances that can mimic or alter the function of hormones in the body. Previous studies have mostly used cell culture to examine EDCs effects on specific hormone responses. We propose to use transgenic zebrafish allowing direct examination of subtle alterations in endocrine organs and hormone responses in living animals to analyze the cellular behavior and gene expression changes that occur following exposure to EDCs. These transgenic zebrafish have the green fluorescent protein (GFP) reporter gene expressed, respectively, in pancreas directed by the insulin promoter, in pituitary by the POMC promoter, and all cells that respond to estrogens by the vitellogenin B1 promoter. Flow cytometry and gene expression analysis by microarrays and microSAGE will be used to provide a profile of the genes activated by EDCs at the cellular level. Cytokines, chemokines, signal transduction messengers, proteases, oncogenes or cell cycle-related products will be the focus studied in the separated GFP-expressing cells and compared between the natural estrogen exposure and xenoestrogen exposure groups. To generate more tools to study endocrine organ development and EDC induced responses, we will also perform a large-scale chemical compound screen using the transgenic embryos from each line. Small molecules that have specific effect in either endocrine cell lineage development or estrogen responses will be selected for future studies. Due to the high conservation of the biological processes to be studied in this proposal, we believe that findings from zebrafish should ultimately help us understand how environmental stresses exert their impact on the human endocrine cell development and responses.