【作者】 孙宏；

【导师】 王心如；

【作者基本信息】 南京医科大学 ， 卫生毒理学， 2008， 博士

【摘要】 环境中存在着大量人工合成的化学物,这些人类创新产物中的一部分可能会干扰人体内分泌系统的正常的功能,导致生殖内分泌系统肿瘤,不孕不育,甚至会影响到人类的生存繁衍,他们属于环境内分泌二扰物（EEDs）。本研究旨在通过建立核受体介导的in vitro报告基因试验方法（也称为转录激活试验）,来研究这些环境化学物对雌激素受体（ER）、雄激素受体（AR）和甲状腺激素受体（TR）功能的干扰作用。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（HRE）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EEDs筛选的有力工具。针对ER、AR和TR三种不同的核受体,我们的研究分为三个部分,分别构建了相应的HRE调控的报告基因质粒,用来瞬时或者稳定转染不同的哺乳动物细胞,并且研究了环境化学物通过核受体介导的内分泌干扰效应。第一部分环境化学物雌激素干扰效应研究目的以虫荧光素酶（Luc）为报告基因,采用瞬时和稳定转染的方法建立ER介导的转录激活试验方法,为化学物拟/抗雌激素活性的检测提供研究工具。应用建立的方法评价烷基酚类（APs）、双酚A（BPA）、苯基苯酚等结构类似物和一些饮用水水源水样本的雌激素活性。方法1.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,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。CV-1细胞转染后用受试化学物染毒,根据该物质能否诱导Luc的表达,判断化学物的ER激动作用。2.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分别共转染MCF-7细胞,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。MCF-7细胞转染后用受试化学物染毒,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。3.采用稳定转染了pVit-tk-Luc质粒的MVLN细胞,接种到96孔板,染毒受试化学物,检测Luc的表达来研究化学物的拟雌激素活性。结果1.rERα报告基因试验方法的实际最低检测限为10¹⁰M E₂,10^-7M达最大值,诱导倍数为溶剂对照的25.9倍,随着E₂染毒剂量的增加,在10^-7M E₂浓度后,Luc的表达量维持在较高的平台水平,EC₅₀为4.7nM。E₂对Luc诱导作用在组内变异系数（CV）为7.8%,组间CV为12.6%。2.hERα报告基因试验方法的实际最低检测限为0.5×10^-10M E₂,10^-9M达最大值,诱导倍数为溶剂对照的17.9倍,随着E₂染毒剂量的加大,在10^-9M E₂浓度后,Luc的量维持在较高的平台水平。EC₅₀为0.16nM。组内CV为6.7%,组间CV为12.8%。3.采用hERα介导的报告系统研究BPA、苯基苯酚和APs等化学物的拟雌激素活性,试验结果表明,随着取代基上碳原子数目从3增加到8,烷基酚及其结构类似化合物诱导Luc表达的能力也在逐步增加,但是所有受试物的拟雌激素活性均远远低于E₂。采用rERα介导的报告系统取得相似的结果,受试化学物的雌激素活性大小是4。丙基酚＜4-丁基酚＜4-戊基酚＜4-苯基苯酚＜4-辛基酚＜BPA。4.采用rERα报告基因系统研究饮用水源地采取的水样品的雌激素干扰活性,结果两个采样点的水样的200倍浓缩品均显示出拟雌激素的活性。5.0.5pM浓度的E2开始诱导MVLN细胞表达Luc,而到了50pM达到最高效应,最大诱导倍数为溶剂对照的4倍。结论1.本研究建立的ER报告基因试验具有较高的灵敏度和重复性。E_@对Luc的诱导是ER依赖性,并呈剂量-效应关系。以上结果表明本研究建立的方法能够有效地筛选ER激动剂和拮抗剂,可作为化学物（抗）雌激素活性筛选的理想模型。2.所研究的APs和BPA等化学物具有拟雌激素活性,其活性随着取代基大小的增加而增强。而且,体外筛选试验所采用的细胞系类型和ER的种属不影响APs及其类似物的雌激素活性的检测。3.所检测的饮用水水源水样本具有拟雌激素活性的能力。4.稳定转染pVit-tk-Luc质粒的MVLN细胞株能用来研究化学物雌激素干扰活性。第二部分环境化学物雄激素干扰效应研究目的采用瞬时和稳定转染方法,建立AR介导的Luc报告基因试验,为化学物拟/抗雄激素活性的检测提供研究工具。应用建立的方法研究一些常用农药和化学物的雄激素干扰活性。方法1.合成含四个ARE和一个TATA启动子的小鼠乳腺癌病毒长末端重复序列（MMTV-ETR）,定向插入报告基因质粒pGL₃-basic的多克隆位点内切酶KpnⅠ和BglⅡ之间,构建受ARE调控的Luc报告基因质粒pMMTV-Luc。将该质粒与表达AR的质粒AR/pcDNA3.1和阳性对照质粒phRL-tk共转染CV-1,建立AR报告基因试验。以5α-双氢睾酮（5α-DHT）为阳性对照检测该方法的筛选效率。CV-1转染后用受试化学物染毒,根据该物质能否诱导Luc的表达,判断化学物的AR激动作用;根据该物质能否拮抗5α-DHT诱导的Luc表达,判断化学物的AR拮抗作用。2.应用稳定转染了pMMTV-Luc质粒的MDA-kb2细胞,接种到96孔板上,单独或者与DHT共同染毒受试化学物,检测Luc表达研究化学物拟/抗雄激素作用。结果1.本方法的最低检测限为10^-10M 5α-DHT。5α-DHT对Luc诱导作用的组内CV为8.9%,组间CV为13.1%。对只转染pMMTV-Luc和phRL-tk而不转染AR/pcDNA3.1的CV-1染毒,5α-DHT不诱导Luc的表达。5α-DHT对共转染三种质粒的CV-1的诱导作用表现为:10^-10M的5α-DHT显著诱导Luc的表达,10^-7M时达最大值,诱导倍数为61.83,随着5α-DHT染毒剂量的加大,在10^-7M-10^-6M范围内Luc的表达量维持在较高的平台水平,计算其半数效应浓度EC₅₀为3.65 nM。糖皮质激素受体（GR）激动剂地塞米松（dexamethasone）不诱导Luc的表达。AR拮抗剂尼鲁他明（nilutamide）和氟鲁他明（flutamide）在10^-7M-10^-5M浓度范围内显著拮抗1nM的5α-DHT诱导的Luc的表达,其半数抑制浓度(IC₅₀)分别为4.05μM和3.49μM。2.在对农药的AR激动作用进行检测时发现氰戊菊酯（Fen）、氯氰菊酯（Cyp）、氯菊酯（Per）以及3-苯氧基苯甲酸（3-PBA）对Luc没有明显的诱导作用。在进行AR拮抗作用检测时发现:Fen、Cyp、Fer以及3-PBA在10^-4M的浓度均能显著拮抗1nM的5α-DHT诱导的Luc的表达。IC₅₀分别为0.37mM、0.42mM、0.43mM和1.21mM。3.双酚A（BPA）、四氯双酚A（TCBPA）和五氯酚（PCP）单独染毒不能的诱导Luc表达。PCP不能拮抗1nM浓度的5α-DHT诱导的Luc的表达。BPA的浓度为10^-7M时对5α-DHT无明显的拮抗作用,随着染毒浓度的加大,在10^-6M和10^-5M时能拮抗1 nM 5α-DHT诱导的Luc的表达,差异有显著性,其IC₅₀为2.14μM。10^-5M浓度的TCB能够显著性抑制1nM 5α-DHT诱导的Luc表达,其IC₅₀为10.45μM。4.10^-10M DHT开始诱导MDA-kb2细胞系表达Luc,而到10^-8M浓度时,作用达到平台期,诱导倍数为对照的9倍左右。结论1.本研究建立的AR报告基因试验具有较高的灵敏度和重复性。5α-DHT对Luc的诱导是AR依赖性,并呈剂量-效应关系。该方法不存在与GR激动剂地塞米松的交叉反应,具有较高的特异性。本方法能够检出AR拮抗剂尼鲁他明和氟鲁他明对5α-DHT作用的拮抗作用。以上结果提示本研究建立的方法能够有效地筛选AR激动剂和拮抗剂,可作为化学物（抗）雄激素活性筛选的理想模型。2.Fen、Cyp、Per和3-PBA无AR激动作用,但他们都能拮抗雄激素的功能。3.BPA、TCBPA和PCP无AR激动作用;BPA和TCBPA能拮抗雄激素功能;PCP未表现出对雄激素的拮抗作用。4.稳定转染了pMMTV-Luc质粒的MDA-kb2细胞株能够用来研究化学物雄激素干扰活性。第三部分环境化学物甲状腺激素干扰效应研究目的建立瞬时转染的TR介导的Luc报告基因试验体系,以及对甲状腺激素反应的GH₃细胞增殖试验,为化学物拟/抗甲状腺激素活性的检测提供研究工具。应用建立的方法研究一些常用农药和化学物的甲状腺激素干扰活性。方法1.采用无血清的PCM培养基配方培养GH₃细胞,在2500细胞/孔的密度种植到96孔板中,检测T₃和受试化学物对细胞增殖的影响。2.将表达人甲状腺激素受体的质粒pCMX-hTRβ和含甲状腺激素反应元件DR4的重组报告基因质粒pDR4-tk-CAT以及内参pSV-β-Gal共同转染到CV-1细胞内,建立hTRβ介导的报告基因试验方法。以T₃作为阳性对照来检测方法的筛选效率。CV-1细胞转染后用受试化学物染毒,根据该物质能否诱导Luc的表达,判断化学物的TR激动作用,根据该物质能否拮抗T₃诱导的Luc表达,判断化学物的TR拮抗作用。3.将表达人TRβ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-L-TRβ与Gal4反应的报告基因质粒pUAS-tk-luc、对照质粒phRL-tk共转染HepG₂细胞,建立TR介导的报告基因试验,以T₃为阳性对照检测方法的筛选效率。CV-1细胞转染后用受试化学物染毒,根据该物质能否诱导Luc的表达,判断化学物的TR激动作用。根据该物质能否拮抗T₃诱导的Luc表达,判断化学物的TR拮抗作用。结果1.1nM浓度的T₃能够诱导相对于溶剂对照3倍的GH₃细胞的表达。OH-PCB₁₀₆和1nM T₃共同染毒GH₃细胞,结果表明:OH-PCB₁₀₆在10^-9M以上浓度时,能够抑制1nM T₃诱导的GH₃细胞的增殖2.采用CAT报告基因研究OH-PCB₁₀₆抗甲状腺激素作用时,10nM T₃能够诱导3倍于对照的CAT的表达。10^-11M浓度的OH-PCB₁₀₆就能够抑制10nM T₃对CAT活性的诱导,随着PCB浓度的增加,这种抑制作用的增加不是很明显,直到增加到10^-5M浓度,才与10^-11M出现显著性下降。3.TR介导的Luc报告基因试验的最低检测限为10^-11M T₃,10^-8M时达最大值,诱导倍数为7.85,随着T₃染毒剂量的加大,在10^-8M-10^-6M范围内Luc的表达量维持在较高的平台水平,计算其半数效应浓度EC₅₀为0.46nM。采用48孔板,10^-9M的T₃对Luc诱导作用组内变异系数为5.9%,组间的变异系数11.7%。4.农药甲萘成和它的代谢产物1-萘酚以及结构类似物2-萘酚三种受试物在10^-4M及以下浓度时,都不能诱导Luc表达,而三者在10^-5M及以上浓度都可以不同程度抑制5nM T₃诱导的报告基因表达,IC₅₀分别为8.40×10^-5M,7.62×10^-5M和7.73×10^-5M。结论1.在体外,OH-PCB₁₀₆具有抑制甲状腺激素活性能力。2.本研究建立的TR介导Luc报告基因试验具有较高的灵敏度和重复性,能够有效地筛选AR激动剂和拮抗剂,可作为化学物（抗）甲状腺激素活性筛选的理想模型。3.甲萘威、1-萘酚和2-萘酚能够拮抗T₃的功能。更多还原

【Abstract】 There were a plenty of man-made chemicals existing in the environment,which attributed to the modem industry.Some of these chemicals might disrupt the normal function of the endocrine system of human being,causing cancer of reproduction organs,even making human lost their fertility.They belonged to the environmental endocrine disruptor（EEDs）.The present study aimed to develop the in vitro nuclear receptor mediated reporter gene assay（also known as transactivation assay）,and investigate the potential disrupting effects of these environmental chemicals to the function of estrogen receptor（ER）, androgen receptor（AR）and thyroid hormone receptor（TR）.On the basic of receptor-mediated theory,the receptor mediated reporter gene assays were designed to identify substances that might interfere with normal hormone activity by acting as an agonist or antagonist through ligand-receptor interaction.The DNA fragment containing hormone responsive element（HRE）was cloned into a reporter gene plasmid, making the reporter gene regulated by the HRE.The reconstructed reporter plasmid and the corresponding expression plasmid of the hormone receptor were co-transfected into a host cell to create an artificial gene expression system.The 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 constructed the reporter plasmids responding to the activation of ER,AR and TR,and then transfected them to the mammalian cell,getting the reporter gene assay systems,by which,the endocrine disrupting effects of some environmental chemicals were assessed.PartⅠEffects of environmental chemicals on the function of estrogen receptorObjectiveIn order to provide a model for screening the（anti）estrogen effects of chemicals,the ER reporter gene assay was developed using the luciferase （Luc）as the reporter gene,by transient and stable transfection.The assay was applied to evaluate the（anti）estrogen effects of some alkylphenol chemicals,bisphenol A,phenylphenol and some samples from water resource.Methods1.The CV-1 was 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.2.The plasmid expressed the protein of Gal4-BD fused human ERαwas constructed and co-transfected into MCF-7 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 E₂.3.MVLN cell was planted into 96 well plates and treated with various concentrations of test chemicals.ER agonist assay was generally performed by quantifying the induction of the reporter gene Luc product Result1.The rERαmediated reporter gene assay could detecte the effects cause by 10^-10M E₂;at the concentration of 10^-7M,the maximal magnitude of the fold induction of 25.9-fold of control was achieved and maintained at a plateau level at the ranges of 10^-7M-10^-6M.The median effective concentration(EC₅₀)value of E₂ was 4.7 nM.The intro coefficient of variation（CV）in 24-well plate was 7.8%,and the inter CV was 12.6%.2.The hERa mediated reporter gene assay could detecte the effects cause by 0.5×10^-10M E₂;at the concentration of 10^-9M,the maximal magnitude of the fold induction of 17.9-fold of control was achieved and maintained at a plateau level at the ranges of 10^-9M-10^-6M.The median effective concentration(EC₅₀)value of E₂ was 0.16 nM.The intro coefficient of variation（CV）in 24-well plate was 6.7%,and the inter CV was 12.8%.3.The estrogenic activity of BPA,phenylphenol and alkylphenols （APs）were assessed by both hERa and rERαreporter assay.The results indicate that,the expression of luciferase increased with each additional carbon in the substituent,from 3 to 8 carbon atoms,but all test alkylphenol chemicals were much less potent than E₂.Briefly,the estrogenicity of test chemicals was 4-propylphenol＜4-tert-butylphenol＜4-pentylphenol＜4-phenylphenol＜4-octylphenol＜BPA.4.Two water samples could induce the rERαmedicated Luc expression.5.0.5pM E2 could induce the MVLN cell to express Luc,and the maximal magnitude of the fold induction of 4-fold of control was achieved at the concentration of 50pM.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.BPA,phenylphenol and APs possessed significant estrogenic activity,and their activity increased with the increase of substituent size in both hERαand rERαassays.Furthermore,the types of cell lines and the sources of ER showed little effect on the estrogenic activity of APs.3.The water sample might disturb the normal function of estrogen.4.MVLN cell had the ability to detect the agonist activity of test chemicals. PartⅡEffects of environmental chemicals on the function of androgen receptorObjectiveIn order to provide a model for screening the（anti）androgen effects of chemicals,the AR reporter gene assay was developed using the Luc as the reporter gene,by transient and stable transfection.The assay was applied to evaluate the（anti）androgen effects of some pesticides and industrial chemicals.Methods1.The oligonucleotides of the mouse mammary tumor virus long terminal repeat（MMTV-LTR）containing four androgen responsive element（ARE）sequences and TATA promoter were inserted into the kpnⅠand BglⅡsites of pGl₃-basic to construct the new plasmid pMMTV-Luc. The CV-1 cell was co-transfected with three plasmids including the expression plasmid AR/pcDNA3.1 containing the cDNA of AR, constructed reporter plasmid pMMTV-Luc containing the Luc regulated by ARE and the control plasmid phRL-tk to create the AR reporter gene assay.The reference androgen 5α-dihydrotestosterone（5α-DHT）was used to verify the performance of the assay.AR agonist activity was generally performed by quantifying the induction of the reporter gene Luc product in response to activation of the AR by the test chemicals.AR antagonism was performed by measure the ability of a test substance to inhibit the induction of the reporter gene Luc product by 5α-DHT.2.MDA-kb2 cell was planted into 96 well plates and treated with various concentrations of test chemicals with or without DHT.Result1.The AR mediated reporter gene assay could detecte the effects cause by 10^-10M 5α-DHT;at the concentration of 10^-7M,the maximal magnitude of the fold induction of 61.83-fold of control was achieved and maintained at a plateau level at the ranges of 10^-7M-10^-6M.The median effective concentration(EC₅₀)value of E₂ was 3.65 nM.The reference androgen 5α-DHT showed no significant effect in inducing Luc activity, when the CV-1 cell was not transfected with AR expression plasmid.The intro CV in 24-well plate was 8.9%,and the inter CV was 13.1%.At the concentration ranges of 10^-7M-10^-5M,nilutamide and flutamide significantly inhibited the Luc activity induced by lnM 5α-DHT.The median inhibitory concentration(IC₅₀)value was 4.05μM and 3.49μM, respectively.2.The three pyrethroid pesticides Fenvalerate（Fen）,Cypermethrin （Cyp）,Permethrin（Per）and their metabolite 3-PBA did not show androgenic transcriptional activity at the tested concentrations.At the concentrations of 10^-4M,Fen,Cyp,Per and 3-PBA significantly inhibited the Luc activity induced by 1nM 5α-DHT,and the IC₅₀value was 0.37 mM,0.42 mM,0.43 mM and 1.21 mM.3.BPA,tetrachlorobisphenol A（TCBPA）pentachlorophenol（PCP） didn’t induce the expression of Luc in AR mediated reporter assay.PCP didn’t inhibit the the Luc activity induced by 1nM 5α-DHT,while BPA and TCBPA could suppress the function of 1nM 5α-DHT,with IC₅₀of 2.14μM and 10.45μM.4.10^-10M DHT could induce the expression of Luc,and the maximal magnitude of the fold induction of 9-fold of control was achieved and maintained at a plateau level at the ranges of 10^-8M-10^-5M. Conclusion1.The assay showed high sensitivity and acceptable repeatability to 5α-DHT.The reference androgen 5α-DHT induced Luc activity in an AR-depended and dose-response manner.In addition,the assay system was highly specific to androgenic compounds without cross-talk to GR agonist.The assay is useful in identifying AR agonists and antagonists from a large number of chemicals.2.Fen,Cyp,Per and 3-PBA could not activate the AR,but all of them showed weak antiandrogenic activity.3.BPA,TCBPA and PCP could not activate the AR;BPA and TCBPA showed antiandrogenic activity while PCP didn’t.4.MDA-kb2 could be used to screen the androgen disruptors.PartⅢEffects of environmental chemicals on the function of thyroid hormone receptorObjectiveIn order to provide a model for screening the（anti）thyroid effects of chemicals,the TR mediated reporter gene assay and GH₃ cell proliferation assay were developed.The assays were applied to evaluate the（anti）thyroid hormone effects of some pesticides and industrial chemicals.Methods1.Rat pituitary tumor cell line GH₃ cell was cultured in serum-free medium PCM.And cells were planted on 96 wells plates at the density of 2500 cells per well and treated with various concentrations of test chemicals.2.The CV-1 was co-transfected with three plasmids including human TRβexpression plasmid pCMX-hTRβ,reporter plasmid pDR4-tk-CAT containing the reporter gene CAT regulated by the thyroid hormone responsive element（TRE）and the control plasmid pSV-β-Gal to develop the hTRβmediated reporter gene assays.The reference thyroid hormone L-3,5,3’-Triiodothyronine（T₃）was used to verify the performance of the assays.TR agonist assay was generally performed by quantifying the induction of the reporter gene CAT product in response to activation of the TRβby the test chemicals.TR antagonist assay was performed by measure the ability of a test substance to inhibit the induction of CAT product inducing by T₃3.The plasmid expressed the protein of Gal4-BD fused human TRβwas constructed and co-transfected into HepG₂ cell with Gal4 responsed reporter plasmid pUAS-tk-luc and the control plasmid phRL-tk to develop the hTRβmediated reporter gene assays.The reference thyroid hormone T₃ and L-Thyroidxin（T₄）was used to verify the performance of the assays.TR agonist assay was generally performed by quantifying the induction of the reporter gene Luc product in response to activation of the TRβby the test chemicals.ER antagonist assay was performed by measure the ability of a test substance to inhibit the induction of Luc product by T₃.Result1.T₃ could stimulate the proliferation of GH₃ cell at 1nM,and the proliferation fold was 3-fold of vehicle contol.When treated the cells together with 1nM T₃,OH-PCB₁₀₆(at concentration of 10^-9M or higher) could suppress the function of T₃.2.10nM T₃ could induce the expression of CAT 3-fold of vehicle control in the hTRβmediated reporter gene assay.At the concentration as low as 10^-11M,OH-PCB₁₀₆could suppress the expression of reporter gene induced by 10nM T₃.However,this kind of suppression didn’t become strong when we increased the concentration of OH-PCB₁₀₆,until the concentration raised to 10^-5M.3.The TRβmediated reporter gene assay could detecte the effects cause by 10^-11M T₃;at the concentration of 10^-8M,the maximal magnitude of the fold induction of 7.85-fold of control was achieved and maintained at a plateau level at the ranges of 10^-8M-10^-6M.The median effective concentration(EC₅₀)value of T₃ was 0.46 nM.The intro CV in 48-well plate was 5.9%,and the inter CV was 11.7%.4.At the concentration below 10^-4M,carbaryl,1-naphthol（1-NAP） and 2-naphthol（2-NAP）couldn’t induce the expression of Luc.However, at or above the concentration of 10^-5M,all of them could inhibit the expression of Luc induced by 5nM T₃,and the IC₅₀was 8.40×10^-5M, 7.62×10^-5M and 7.73×10^-5M for carbaryl,1-NAP and 2-NAP.Conclusion1.OH-PCB₁₀₆showed anti-thyoid hormone activity.2.The assay showed high sensitivity and acceptable repeatability to T₃ and T₄.The reference thyroid hormone T₃ induced Luc activity in a TR-depended and dose-response manner.The assay is useful in identifying TR agonists and antagonists from a large number of chemicals.3.Carbaryl,1-NAP and 2-NAP might disrupt the normal function of T₃.更多还原