【作者】 陈春波；

【导师】 薛整风； 李湘鸣； 李厚达；

【作者基本信息】 扬州大学 ， 动物学， 2006， 硕士

【摘要】 亲电性化学物是指在环境污染物中一大类可与细胞生物大分子上的亲核中心相结合或可通过生物转化相结合的化合物,除可以引起急、慢性中毒外,还可引起肿瘤。亲电性化学物是环境中的主要污染物,所以越来越引起环境科学研究者的高度关注。环境污染物多数为亲电性（Electrophile）化学物质,如TCDD,CdCl₂,多氯联苯,HgCl₂,Pb（C₂H₃O₂）₂等。国外许多学者研究发现[1-6],在哺乳动物细胞中,具有与亲电性化学物发生反应的特异DNA基序（Motif）,称为亲电性物质反应元件（Electrophile Response Element,EPRE）,这一基因的调控区基序是非常保守的,调控下游有关基因的表达。EPRE可介导多种与机体抗氧化有关的基因的表达,如γ-谷氨酰半胱氨酸合成酶基因,半胱氨酸谷胱甘肽转移酶基因等,其酶活性的表达程度与亲电性化学污染物对EPRE激活程度有关,两者间存在剂量反应关系。本研究根据细胞双杂交原理,分别从p（Gal4）5-TK/CAT和pMT/V5-His扩增TK（胸腺嘧啶）和MT（金属硫蛋白）基因基本启动子,分别与虫荧光素酶报告基因（LUC）相连,将人工合成的亲电性化学反应元件（EPRE）插入LUC的上游,构建成亲电性化学物调控的LUC报告载体（pEPRE-TK-LUC和pEPRE-MT-LUC）。用脂质体将pERRE-TK-LUC和pEPRE-MT-LUC分别转染于HeLa细胞和昆虫细胞（Sf9）。用瞬时表达法发现重组HeLa细胞荧光素酶与亲电性化学物氯化汞、氯化镉和亚砷酸钠具有剂量效应关系,而重组Sf9与亲电性化学物无剂量效应关系,这可能是Sf9对亲电性化学物质的毒性特别敏感。用G418对重组HeLa细胞进行筛选,阳性细胞经顺丁烯乙酸二乙酯（DEM）实验,发现荧光素酶与DEM间存在剂量效应关系。然而,在正常的pH条件下,底物D-荧光素不能进入细胞,在测定荧更多还原

【Abstract】 Electrophile chemical is a large class of environmental pollutants which can combine with nucleophilic center of large molecules directly in cell biology or bind the center by bio-transformation to produce biological compounds. This chemical can not only cause acute and chronic poisoning, but also cause cancer. Electrophile chemical is the major pollutant in the environment, this is very concerned by many of environmental scientists.Most of environmental pollutants are electrophile chemicals, such as TCDD,CdCl₂, HgCl₂,Pb（C₂H₃O₂）₂ and so on. Many foreign researchers have found out that some special motifs in the genes of Mammals cells, which can specifically respond to electrophile chemicals. This motif is called Electrophile Response Element（EPRE）. The region of the motif is very conservative, which regulates the expresssion of downstream reporter gene. The antioxidant genes such as r-glutamylcysteine synthetase and cysteineglutathione transferase gene are mediated by EPRE. The degrees of the gene expression have some relationships with EPRE activation degrees caused by electrophile chemicals. The dose-effect model exists between them.TK（thymidine kinase） and MT（metallothionein） minimal promoter was selected from the plasmid of p（Gal4）5-TK/CAT and pMT/v5-his according to the pricinple of mammalian cell two hybrid.The two promoters were ligated with luciferase reporter gene respectively,and then the artificial electrophile response element （EPRE ） were inserted into the upstream of luciferase reporter gene.In this way,The luciferase reporter更多还原