【作者】 徐小林；

【导师】 李忠； 王心如；

【作者基本信息】 南京医科大学 ， 卫生毒理学， 2008， 硕士

【摘要】 雌激素受体报告基因试验也称雌激素受体转录激活试验,是EPA推荐的用于环境雌激素筛选的in vitro方法之一。雌激素受体报告基因试验是以受体介导理论为基础,应用基因重组技术,将报告基因置于外源性激素反应元件（ERE）的调控之下,构建重组报告基因载体,应用基因转染技术导入真核细胞内,通过检测化学物作用下报告基因编码的酶活性或蛋白表达的变化,间接反映内源性基因的表达情况。这种方法既可检测化学物与受体的结合能力,又可检测结合后引起的生物学效应,而且能够区分激动剂和拮抗剂,与受体结合试验相比可提供更多的信息,因此成为环境雌激素筛选的有力工具。本研究构建了两种不同ERα介导的报告基因试验,并探讨了它们对三种拟除虫菊酯类农药的雌激素活性的影响。第一部分雌激素受体介导的报告基因试验方法的建立目的建立人ERα和大鼠ERα介导的报告基因试验体系,并比较这两种方法的反应性和灵敏度。方法1.将表达人ERα配体结合域和Gal4-BD（酵母转录因子结合域）融合蛋白的质粒pGal4-ERαdef与Gal4反应的报告基因质粒pUAS-tk-luc、对照质粒phRL-tk分别转染CV-1细胞,转染方式为瞬时转染,建立ER介导的报告基因试验。以E₂为阳性对照检测方法的筛选效率。2.将表达大鼠ERα的质粒rERα/pCI与重组Luc报告基因质粒pERE-aug-Luc、对照质粒phRL-tk共转染CV-1细胞,转染方式为瞬时转染,建立rERα介导的报告基因试验。以雌二醇（E₂）为阳性对照检测方法的筛选效率。结果1.在两种ERα体系中,E₂能明显的诱导Luc的表达,并呈显著的剂量-反应关系,10^-7M达到最大值,最大诱导倍数分别为19.6和21.6倍,EC₅₀为2.4 nM和0.9 nM。2.用浓度从10^-10M到10^-8M的DES染毒hERα体系时,Luc的表达量与E₂相似,且10μM的ICI182,780能完全阻断这一效果。结论1.本研究建立的两种ERα报告基因试验具有较高的灵敏度和重复性。E₂对Luc的诱导是ER依赖性的,并呈剂量-效应关系。表明该实验系统能够用来检测拟雌激素活性的物质。2.在hERα体系中,ICI182,780能有效抑制E₂诱导的Luc的表达,表明hERα体系可以用来检测化学物的抗雌激素活性。3.两种ERα对报告基因试验E₂的反应性无明显区别,两种体系的灵敏度也相似。第二部分雌激素受体介导的报告基因试验方法的应用目的应用两种雌激素受体介导的报告基因试验体系检测三种拟除虫菊酯类农药的拟或（抗）雌激素活性,并比较它们对这几种化学物的反应性和灵敏度。方法1.CV-1细胞转染pUAS-tk-luc、pGal4-ERαdef和phRL-tk质粒后用三种拟除虫菊酯类农药（氰戊菊酯,氯氰菊酯,苄氯菊酯）染毒,根据该物质能否诱导Luc的表达,判断化学物的ER激动作用。根据该物质能否拮抗E₂诱导的Luc表达,判断化学物的ER拮抗作用。2.CV-1细胞转染rERα/pCI、pERE-aug-Luc和phRL-tk质粒后用三种拟除虫菊酯类农药染毒,根据该物质能否诱导Luc的表达,判断化学物的ER激动作用。结果1.三种拟除虫菊酯类农药在两种体系中可以诱导Luc的表达,并呈显著的剂量-反应关系,在10^-4M达到最大值。2.三种拟除虫菊酯类农药均无法抑制10^-9M E₂所诱导的Luc表达。结论1.所研究的三种拟除虫菊酯类农药都具有一定的拟雌激素活性,但活性强度均远远低于E₂。2.三种拟除虫菊酯类农药均无抗雌激素活性。3.两种体系对化学物的反应性和灵敏度基本相同,即两种ERα对报告基因试验筛选雌激素活性物质无明显区别。更多还原

【Abstract】 The estrogen receptor mediated reporter gene assay,also named receptor transcriptional activation assay,is an in vitro methods to screen the environmental estrogens proposed by the US EPA.On the basic of receptor-mediated theory,the estrogen receptor mediated reporter gene assays were designed to identify substances that might interfere with normal estrogenic activity by acting as an agonist or antagonist through ligand-receptor interaction.The DNA fragment containing estrogen responsive element（ERE）was cloned into a reporter gene plasmid, making the reporter gene regulated by the ERE.The reconstructed reporter plasmid and the corresponding expression plasmid of the estrogen receptor were co-transfected into a host cell to create an artificial gene expression system.The estrogen receptor mediated reporter gene assay provided a relatively simple way to indirectly reveal whether a substance could activate or inhibit the transcriptional activation of receptor-regulated genes by the measurement of the reporter gene product, typically an enzyme or a protein.The assays possessed some advantages over the receptor binding assays because they provided the information on both the ability of receptor binding and the biological response after binding（i.e.,RNA transcription）.In addition,unlike receptor binding assays,the assays could distinguish a receptor agonist from an antagonist. In this study,we conducted two different types of ERαmediated reporter gene assays in African green monkey kidney cell line CV-1 to clarify the relationship between these two assays for three pyrethroids.PartⅠDevelopment of estrogen receptor mediated reporter gene assaysObjectiveTwo ER reporter gene assays employing the human ERαand rat ERαwas developed in order to evaluate the（anti）estrogen effects of chemicals,and the assays was applied to clarify the relationship between these two different types of ERα.Methods1.The plasmid expression Gal4-BD fused human ERαwas constructed and transiently co-transfected into CV-1 cell with Gal4 responsed reporter plasmid pUAS-tk-luc and the control plasmid phRL-tk to develop the hERαmediated reporter gene assays.The reference estrogen E₂ was used to verify the performance of the assays.ER agonist assay was generally performed by quantifying the induction of the reporter gene Luc product in response to activation of the ERαby the test chemicals.ER antagonist assay was performed by measure the ability of a test substance to inhibit the induction of the reporter gene Luc product by E₂2.The CV-1 was transiently co-transfected with three plasmids including rat ERαexpression plasmid rERα/pCI,reporter plasmid pERE-aug-Luc containing the reporter gene Luc regulated by the estrogen responsive element（ERE）and the control plasmid phRL-tk to develop the rERαmediated reporter gene assays.The reference estrogen 17β-estradiol（E₂）was used to verify the performance of the assays.ER agonist assay was generally performed by quantifying the induction of the reporter gene Luc product in response to activation of the ERαby the test chemicals.Result1.Following a 24-h exposure to E₂,the luciferase activity in these two reporter gene assays was stimulated in a dose-dependent manner at concentrations of more than 10^-11M.The maximal induction of 19.6-fold and 21.6-fold of vehicle controls were obtained at 10^-7M,with EC₅₀ values of 2.4nM and 0.9nM respectively.2.In hERαreporter assay,the potent synthetic estrogen,DES was evaluated at concentrations from 10^-10M to 10^-8M.As expected,it induced luciferase activity at concentrations similar to that seen with E₂.And the ER antagonist ICI182,780 could completely inhibit the response to E₂ in this assay.Conclusion1.The assays showed high sensitivity and acceptable repeatability to E₂.The reference estrogen E₂ induced Luc activity in an ER-depended and dose-response manner.The assays were useful in identifying ER agonists from a large number of chemicals.2.The results revealed that hERαreporter assay could be adapted for evaluation of the antiestrogenic activity of diverse synthetic chemicals.3.The two assays showed the similar reliability and responsiveness.PartⅡApplication of estrogen receptor mediated reporter gene assaysObjectiveTwo reporter gene assays employing the human ERαand rat ERαwas applied to evaluate the（anti）estrogen effects of three frequently encountered pyrethroid insecticides and clarify the relationship between these two different types of ERα. Methods1.The reporter plasmid pUAS-tk-luc、pGal4-ERαdef and the control plasmid phRL-tk was transiently co-transfected into CV-1 cell for screening the（anti）estrogenic activity of three pyrethroid insecticides.ER agonist assay was generally performed by quantifying the induction of the reporter gene Luc product in response to activation of the ER by the test chemicals.ER antagonist assay was performed by measure the ability of a test substance to inhibit the induction of the reporter gene Luc product by E₂.2.The reporter plasmid rERα/pCI、pERE-aug-Luc and the control plasmid phRL-tk was transiently co-transfected into CV-1 cell to screen the（anti）estrogenic activity of three pyrethroid insecticides.ER agonist assay was generally performed by quantifying the induction of the reporter gene Luc product in response to activation of the ER by the test chemicals.Result1.Three pyrethroid insecticides（cypermethrin,fenvalerate, permethrin）increased the luciferase expression in a dose-dependent manner,and the maximum induction was observed at a dose of 10^-4M in both assays.2.In hERαreporter assay,the tested pesticide was added to the assay system along with 1 nM of E₂ in the medium.As a result,none of the three test chemicals could reduce the E₂-inducing luciferase expression in this assay. Conclusion1.All of the three pyrethroids possessed estrogenic activities,but they were much less potent than E₂.2.None of the three pyrethroids exhibited statistically significant antiestrogenic activity in hERαgene assay.3.No marked differences in the estrogenic activities of pyrethroids were found between these two assays.更多还原