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

本研究旨在分析药物代谢的主要氧化酶细胞色素P4501A1对核受体芳香烃受体(AhR)转录激活的基因表达调控机制。1.修饰物克隆细胞系的建立。将人肝癌细胞株HepG2和小鼠肝癌细胞株Hepa1c1c7分别与小鼠CYP1A1启动子连接的绿色荧光蛋白报告基因载体导入，筛选出稳定的转化子。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AhR依赖转录的影响。我构建了一个独特的IRES构建载体，并利用该载体，发现在人的CYP1A1基因的内部基因组区域，特别是在第一内含子的内部，存在一个能抑制AhR-TCDD诱导的CYP1A1基因转录的有效序列。3.利用随机杂合核酶文库筛选小鼠肝癌细胞对苯并[1]并[1]的抗性调控基因。将随机杂合核酶文库导入上述建立的绿色荧光蛋白报告细胞系(1)。在这些转染组中，用细胞分选机分离出3-甲基环丙烷高表达的一组绿色荧光蛋白，然后收集这些细胞的文库。

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.