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Molecular mechanism of intracellular action of modifiers modulating the susceptibility to chemical exposure.

调节化学暴露敏感性的修饰剂的细胞内作用的分子机制。

基本信息

批准号：
17580274
负责人：
OHSAKO Seiichiroh
金额：
$ 2.3万
依托单位：
The University of Tokyo
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

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17580274/
关键词：
CYP1A1 AhR EGFP Reporter Hepatome

项目摘要

This study was aimed at analyzing about gene expression regulation mechanism of cytochrome P4501A1(CYP1A1), the major drug metabolism oxidizing enzyme, in aspect of with the base level, IC50, or maximum induction, regarding the transcription activation of nuclear receptor aryl-hydrocarbon receptor(AhR).1.Establishment of cell-lines for the modifier cloning. Human hepatoma HepG2 or mouse hepatoma Hepa1c1c7 was transfected with a EGFP reporter construct which was connected a mouse CYP1A1promoter, and then stable tranformants was selected. These cell-lines can monitor an AhR dependence transcription induction of CYP1A1 gene by fluorescence measurement.2.Analysis of influence of the first intron on an AhR dependence transcription of human CYP1A1 gene by using IRES connected luciferase system. I generated an unique IRES construct vector, and by using it, then revealed that there were an effective sequence which can inhibit the induction of CYP1A1gene transcription by AhR-TCDD, in the internal genomic region of human CYP1A1 gene, especially inside of the first intron.3.A screening of genes which modulate benzopyrene resistance of the mouse hepatoma Hepa1c1c7 by using randomized hybrid ribozyme library. A randomized hybrid ribozyme library was introduced to the EGFP-reporter cell line established above(1). Among these transfected cell groups, a high group of EGFP expression strength by 3-methylchorantrene were separated with a cell-sorter machine and then collected the library from these cells.

本研究旨在从基础水平、IC50或最大诱导等方面分析主要药物代谢氧化酶细胞色素P4501A1（CYP1A1）对核受体芳基烃受体（AhR）转录激活的基因表达调控机制。 1.修饰克隆细胞系的建立。用连接小鼠CYP1A1启动子的EGFP报告基因转染人肝癌HepG2或小鼠肝癌Hepa1c1c7，然后选择稳定的转化子。这些细胞系可以通过荧光测量监测CYP1A1基因的AhR依赖性转录诱导。2.利用IRES连接的荧光素酶系统分析第一内含子对人CYP1A1基因的AhR依赖性转录的影响。我构建了一个独特的IRES构建载体，并通过使用它，发现在人CYP1A1基因的内部基因组区域，特别是第一个内含子内部，存在一个可以抑制AhR-TCDD诱导CYP1A1基因转录的有效序列。3.利用随机杂交筛选调节小鼠肝癌Hepa1c1c7苯并芘抗性的基因核酶文库。将随机杂合核酶文库引入上述建立的 EGFP 报告细胞系 (1)。在这些转染的细胞组中，用细胞分选机分离出3-甲基chorantrene的高EGFP表达强度组，然后从这些细胞中收集文库。