高通量筛选环境雌激素类化合物的方法学研究
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摘要
环境内分泌干扰物是指由于人类的生产和生活活动释放到环境中的、对人体内和动物体内内源性的正常激素功能施加影响，从而影响机体内分泌系统化学物质作用的物质，这类物质通称为“外源性干扰内分泌的化学物质”。环境雌激素是环境内分泌干扰物中最大的一类，也是研究最多，最受重视的一类。目前，检测环境雌激素的方法主要有化学法和生物法。化学法检测环境雌激素灵敏度高、特异度好，定性和定量准确。但是用仪器检测环境雌激素，只能对已知的环境雌激素进行检测，而且，在检测前一般还要对样品要进行提取、净化、浓缩等前期处理。由于这些化学方法使用的仪器昂贵、检测成本高、分析过程冗长、技术复杂、对检测人员专业化程度要求高，不适合大量样本的环境雌激素常规检测。建立在生物学基础上检测环境雌激素的方法大多需要进行细胞培养，对实验室条件要求较高、费时费力，不同的实验室使用的细胞不同，不便于结果的相互比较，培养细胞所用的血清也要经过活性炭和葡聚糖处理，去除血清中内源性的雌激素后才能使用，否则会出现假阳性的结果。有的环境雌激素还具有细胞毒性，这样有可能导致得出错误的结论。
因此，根据大多数环境雌激素与机体作用都是干扰天然雌激素与雌激素受体结合，我们以雌激素受体竞争结合分析为基础，建立以下三种方法来高通量的筛选环境雌激素。
第一部分基于ELISA的受体竞争结合法检测环境雌激素目的：环境雌激素是最主要的一类环境内分泌干扰物，它们进入生物体后，可通过干扰生物体自身激素的合成、分泌、转运、结合、活性反应、代谢、消解或产生类似生物体自身激素的作用，对生物有机体维护正常的动态平衡、繁殖、生长及行为产生不利的影响。本研究的目的旨在建立环境雌激素的体外快速筛选方法。方法：采用免疫测定介导受体竞争结合试验定量检测环境雌激素。将17β-雌二醇-BSA固定在微孔板上，环境雌激素与微孔板上的17β-雌二醇-BSA竞争性的与雌激素受体结合，与微孔板上17β-雌二醇-BSA结合的受体与雌激素受体的抗体、雌激素受体抗体的二抗形成一个三明治形式的复合物，通过显色剂显色，最后用比色法对试验结果进行定量。结果：整个反应中环境雌激素浓度越高，溶液的吸光度值的变化就越小，环境雌激素的浓度与吸光度值成负相关。定量研究可以得到环境雌激素剂量一效应关系曲线，在一定浓度范围内(10ng/L～100μg/L)，溶液的吸光度值与环境雌激素的浓度直接呈现良好的线形关系(R~2=0．9583)。利用这种方法能检测到的标准品雌二醇的最低浓度为10ng/L。结论：本方法检测环境雌激素操作简便、无需复杂昂贵的仪器，具有广泛的应用前景。
第二部分荧光标记17β-雌二醇检测环境雌激素目的：荧光标记这种非放射性标记技术已成为现代分析检测中不可或缺的重要方法之一，然而将此技术应用于环境雌激素的筛选并不广泛。本研究旨在探索荧光标记技术在环境雌激素定量检测中的应用。方法：首先将雌激素受体的抗体固定于微孔板中，将待测样本或17β-雌二醇、与定量的17β-雌二醇-FITC、雌激素受体混和后加入微孔板中，孵育后取上清(上清中含有因环境雌激素干扰而未能与雌激素受体结合的的17β-雌二醇-FITC)，加入另一新的微孔板中，用酶标仪检测溶液中荧光信号的强度。结果：溶液的荧光强度与环境雌激素的浓度呈正相关，在一定浓度范围内(10ng/L～100μg/L)，溶液的荧光强度与环境雌激素浓度之间呈现良好的线性关系(R~2=0．9793)。利用这种方法能够检测到的标准品17β-雌二醇的最低浓度为10 ng/L。结论：本研究方法检测环境雌激素灵敏度高，无需复杂昂贵的设备，试验操作非常简单，可在微孔板和芯片上进行，是一种高通量快速筛选环境雌激素的方法。
第三部分纳米金颗粒修饰雌激素受体反应原件检测痕量
环境雌激素的方法学研究目的：雌激素受体竞争结合分析试验是重要的环境雌激素的筛选试验，是美国EDSTAC首选的第一级环境内分泌干扰物筛选试验之一。本研究根据环境雌激素在机体内发挥作用的基本机理，即环境雌激素进入机体，结合并激活雌激素受体，进而使受雌激素受体控制的基因表达。本研究是利用纳米金标记检测技术介导雌激素受体竞争结合分析建立的一种环境雌激素高通量快速筛选方法。方法：首先将17β-雌二醇-BSA固定于微孔板中，将待测样本与定量的雌激素受体混和后加入微孔板中，孵育后取上清(上清中含有因环境雌激素干扰而未能与微孔板上的17β-雌二醇结合的雌激素受体)，将上清加入另一包被有雌激素受体抗体的的微孔板中，使雌激素受体与抗体结合，然后加入标记有纳米金的雌激素反应元件探针，孵育一段时间，用银染增强显色检测光密度。结果：在一定的反应时间内，溶液的吸光度值与环境雌激素的量呈正相关，定量研究可以得到在一定反应时间点(银染增强时间100～140秒)和一定浓度范围内(100pg/L～1μg/L)，溶液的吸光度值与17β-雌二醇之间呈现良好的线性关系(R~2=0．9764)。利用这种方法能够检测到的标准品17β-雌二醇的最低浓度为100 pg/L。结论：本研究方法检测环境雌激素灵敏度高，无需复杂昂贵的设备，操作简单，可在微孔板和芯片上进行，是一种高通量环雌激素快速筛选方法，与以往的方法相比，灵敏度可提高2～3个数量级。
Endocrine disrupting compounds(EDCs)are a defined category ofenvironmental contaminants,which interfere with endocrine system functionand persistent in the environment.A large part of EDCs have been identified thatinduce estrogen-like responses and are classified as estrogenic EDCs.Currentdetection methods for estrogenic EDCs include chemical analytical methods andbiologically based assays.The chemical analytical methods provide excellentsensitivity and precision for monitoring environmental estrogen.However,thesetechniques only measure specific estrogenic EDCs individually,so the targetcompound must have already been identified as have estrogenic properties.Thechemical analytical methods are costly and time-consuming,since they needcomplicated steps for sample preparation,expensive equipments and highlyskilled technicians,which will not be adapt for large numbers of samples toscreened.Biologically based assays always based on Cell culture.Serum used incell culture must be treated by charcoal and dextran T70 to remove the sexsteroids.Different laboratories using different cell lines and the result can not becompared.Cellular assays are also confronted with the problem of cytotoxicsubstances,which may be present in environmental samples and could lead to erroneous results unless proper controls are included.
Ligand binding to the estrogen receptor is the initial step for mostenvironmental estrogen entered the body.According to this mechanism,weestablished three method to screen the environmental estrogen.
PartⅠCompetitive Recombinant Estrogen Receptor-based ELISAfor Detection of Estrogen in Environment
Objective Environmental estrogen is a type of EDCs which can interfere withthe synthesis,secretion,transport,binding,action,or elimination of naturalhormones in vivo.This study aimed at developing a rapid and sensitive bioassayfor the detection of environmental estrogens.Methods Here we present asimple method for quantitative assessment of environmental estrogens based oncompetitive estrogen receptor(ER)mediated immunoassay in microplates,inwhich estrogen,ER,anti-ER antibody and secondary antibody labeledhorseradish peroxidase(HPR)can form a sandwich complex.Results Theassay result showed that as little as 10ng/L of 17β-estradiol could be detectedand have a linear range from 10ng/L to 100μg/L(R~2=0.9583).ConclusionThis method provides a simple way to quantification environmental estrogens insamples.
PartⅡApplication of fluorescent labeling 17β-estradiolin the detection of environmental estrogen
Objictive:The fluorescent labeling is one of the most important methods inmodem analysis field as a non-radioactive labeling technique.However,it hasnot been widely applied in environmental estrogen screening.This study aimedat exploring the application of fluorescent labeling technology in the field of environmental estrogens.Method:Anti-ER is immobilized on the blackmicroplates.In the second step(competition step)the environmental sample isincubated together with a limited amount of 17β-estradiol-FITC conjugate andER.After the receptor binding reaction,the supernatant,which contains17β-estradiol-FITC which did not bind with the ER is shifted into another blackmicroplate.Finally the signals are detected with a microplate reader.ResultsThe assay result showed that as little as 10ng/L of 17β-estradiol could bedetected and have a linear range from 10ng/L to 100μg/L(R~2=0.9793).Conclusion The fluorescent labeling technology is a rapid and cost-effectivemethod to screen environmental estrogen.
PartⅢDensitometry Determination of Estrogenic EDCs Using
Gold Nanoparticle-Modified Estrogen Response Element ProbesObjective Estrogen receptor binding assay is an important approach to screenenvironmental estrogens.It is preferred as Tier 1 Screening method forendocrine disruptors by EDSTAC.In order to rapid screen environmentalestrogens,we develop a new high-throughout screening method based onnanoparticle technology.Since estrogen receptor(ER)is a ligand-independentreceptor,we design a simple competitive binding assay in which environmentalestrogens in the ample compete with natural estrogen(17β-estradiol)for bindingto ER.Methods 17β-estradiol-BSA conjugate is immobilized on themicroplates.In the second(competition)step the environmental sample isincubated together with a limited amount of ER.After the receptor bindingreaction,the supernatant,which contains environmental estrogen-ER complex,is incubated in another microplate coated with anti-ER antibody.Because ERhomodimerization can bind to specific sites on DNA- estrogen response elements(EREs),Nanoparticle -modified ERE probes are added and incubated.Finally the signals are amplified by silver enhancement and recorded withabsorbance.The intensity of signal is proportional to the quantity ofenvironmental estrogen in the sample.Results The assay result showed that aslittle as 100pg/L of 17β-estradiol could be detected and have a linear range from100pg/L to 1μg/L(R~2=0.9764).Conclusion The gold nanoparticle -modifiedERE assay is a screening method for the detection of environmental estrogenswith reliable,low cost,rapid,high-throughout and could be performed onmicroplates or chips.Compared with the existing methods,the detection limitcan be improved 2-3 orders of magnitude in this assay.
因此，根据大多数环境雌激素与机体作用都是干扰天然雌激素与雌激素受体结合，我们以雌激素受体竞争结合分析为基础，建立以下三种方法来高通量的筛选环境雌激素。
第一部分基于ELISA的受体竞争结合法检测环境雌激素目的：环境雌激素是最主要的一类环境内分泌干扰物，它们进入生物体后，可通过干扰生物体自身激素的合成、分泌、转运、结合、活性反应、代谢、消解或产生类似生物体自身激素的作用，对生物有机体维护正常的动态平衡、繁殖、生长及行为产生不利的影响。本研究的目的旨在建立环境雌激素的体外快速筛选方法。方法：采用免疫测定介导受体竞争结合试验定量检测环境雌激素。将17β-雌二醇-BSA固定在微孔板上，环境雌激素与微孔板上的17β-雌二醇-BSA竞争性的与雌激素受体结合，与微孔板上17β-雌二醇-BSA结合的受体与雌激素受体的抗体、雌激素受体抗体的二抗形成一个三明治形式的复合物，通过显色剂显色，最后用比色法对试验结果进行定量。结果：整个反应中环境雌激素浓度越高，溶液的吸光度值的变化就越小，环境雌激素的浓度与吸光度值成负相关。定量研究可以得到环境雌激素剂量一效应关系曲线，在一定浓度范围内(10ng/L～100μg/L)，溶液的吸光度值与环境雌激素的浓度直接呈现良好的线形关系(R~2=0．9583)。利用这种方法能检测到的标准品雌二醇的最低浓度为10ng/L。结论：本方法检测环境雌激素操作简便、无需复杂昂贵的仪器，具有广泛的应用前景。
第二部分荧光标记17β-雌二醇检测环境雌激素目的：荧光标记这种非放射性标记技术已成为现代分析检测中不可或缺的重要方法之一，然而将此技术应用于环境雌激素的筛选并不广泛。本研究旨在探索荧光标记技术在环境雌激素定量检测中的应用。方法：首先将雌激素受体的抗体固定于微孔板中，将待测样本或17β-雌二醇、与定量的17β-雌二醇-FITC、雌激素受体混和后加入微孔板中，孵育后取上清(上清中含有因环境雌激素干扰而未能与雌激素受体结合的的17β-雌二醇-FITC)，加入另一新的微孔板中，用酶标仪检测溶液中荧光信号的强度。结果：溶液的荧光强度与环境雌激素的浓度呈正相关，在一定浓度范围内(10ng/L～100μg/L)，溶液的荧光强度与环境雌激素浓度之间呈现良好的线性关系(R~2=0．9793)。利用这种方法能够检测到的标准品17β-雌二醇的最低浓度为10 ng/L。结论：本研究方法检测环境雌激素灵敏度高，无需复杂昂贵的设备，试验操作非常简单，可在微孔板和芯片上进行，是一种高通量快速筛选环境雌激素的方法。
第三部分纳米金颗粒修饰雌激素受体反应原件检测痕量
环境雌激素的方法学研究目的：雌激素受体竞争结合分析试验是重要的环境雌激素的筛选试验，是美国EDSTAC首选的第一级环境内分泌干扰物筛选试验之一。本研究根据环境雌激素在机体内发挥作用的基本机理，即环境雌激素进入机体，结合并激活雌激素受体，进而使受雌激素受体控制的基因表达。本研究是利用纳米金标记检测技术介导雌激素受体竞争结合分析建立的一种环境雌激素高通量快速筛选方法。方法：首先将17β-雌二醇-BSA固定于微孔板中，将待测样本与定量的雌激素受体混和后加入微孔板中，孵育后取上清(上清中含有因环境雌激素干扰而未能与微孔板上的17β-雌二醇结合的雌激素受体)，将上清加入另一包被有雌激素受体抗体的的微孔板中，使雌激素受体与抗体结合，然后加入标记有纳米金的雌激素反应元件探针，孵育一段时间，用银染增强显色检测光密度。结果：在一定的反应时间内，溶液的吸光度值与环境雌激素的量呈正相关，定量研究可以得到在一定反应时间点(银染增强时间100～140秒)和一定浓度范围内(100pg/L～1μg/L)，溶液的吸光度值与17β-雌二醇之间呈现良好的线性关系(R~2=0．9764)。利用这种方法能够检测到的标准品17β-雌二醇的最低浓度为100 pg/L。结论：本研究方法检测环境雌激素灵敏度高，无需复杂昂贵的设备，操作简单，可在微孔板和芯片上进行，是一种高通量环雌激素快速筛选方法，与以往的方法相比，灵敏度可提高2～3个数量级。
Endocrine disrupting compounds(EDCs)are a defined category ofenvironmental contaminants,which interfere with endocrine system functionand persistent in the environment.A large part of EDCs have been identified thatinduce estrogen-like responses and are classified as estrogenic EDCs.Currentdetection methods for estrogenic EDCs include chemical analytical methods andbiologically based assays.The chemical analytical methods provide excellentsensitivity and precision for monitoring environmental estrogen.However,thesetechniques only measure specific estrogenic EDCs individually,so the targetcompound must have already been identified as have estrogenic properties.Thechemical analytical methods are costly and time-consuming,since they needcomplicated steps for sample preparation,expensive equipments and highlyskilled technicians,which will not be adapt for large numbers of samples toscreened.Biologically based assays always based on Cell culture.Serum used incell culture must be treated by charcoal and dextran T70 to remove the sexsteroids.Different laboratories using different cell lines and the result can not becompared.Cellular assays are also confronted with the problem of cytotoxicsubstances,which may be present in environmental samples and could lead to erroneous results unless proper controls are included.
Ligand binding to the estrogen receptor is the initial step for mostenvironmental estrogen entered the body.According to this mechanism,weestablished three method to screen the environmental estrogen.
PartⅠCompetitive Recombinant Estrogen Receptor-based ELISAfor Detection of Estrogen in Environment
Objective Environmental estrogen is a type of EDCs which can interfere withthe synthesis,secretion,transport,binding,action,or elimination of naturalhormones in vivo.This study aimed at developing a rapid and sensitive bioassayfor the detection of environmental estrogens.Methods Here we present asimple method for quantitative assessment of environmental estrogens based oncompetitive estrogen receptor(ER)mediated immunoassay in microplates,inwhich estrogen,ER,anti-ER antibody and secondary antibody labeledhorseradish peroxidase(HPR)can form a sandwich complex.Results Theassay result showed that as little as 10ng/L of 17β-estradiol could be detectedand have a linear range from 10ng/L to 100μg/L(R~2=0.9583).ConclusionThis method provides a simple way to quantification environmental estrogens insamples.
PartⅡApplication of fluorescent labeling 17β-estradiolin the detection of environmental estrogen
Objictive:The fluorescent labeling is one of the most important methods inmodem analysis field as a non-radioactive labeling technique.However,it hasnot been widely applied in environmental estrogen screening.This study aimedat exploring the application of fluorescent labeling technology in the field of environmental estrogens.Method:Anti-ER is immobilized on the blackmicroplates.In the second step(competition step)the environmental sample isincubated together with a limited amount of 17β-estradiol-FITC conjugate andER.After the receptor binding reaction,the supernatant,which contains17β-estradiol-FITC which did not bind with the ER is shifted into another blackmicroplate.Finally the signals are detected with a microplate reader.ResultsThe assay result showed that as little as 10ng/L of 17β-estradiol could bedetected and have a linear range from 10ng/L to 100μg/L(R~2=0.9793).Conclusion The fluorescent labeling technology is a rapid and cost-effectivemethod to screen environmental estrogen.
PartⅢDensitometry Determination of Estrogenic EDCs Using
Gold Nanoparticle-Modified Estrogen Response Element ProbesObjective Estrogen receptor binding assay is an important approach to screenenvironmental estrogens.It is preferred as Tier 1 Screening method forendocrine disruptors by EDSTAC.In order to rapid screen environmentalestrogens,we develop a new high-throughout screening method based onnanoparticle technology.Since estrogen receptor(ER)is a ligand-independentreceptor,we design a simple competitive binding assay in which environmentalestrogens in the ample compete with natural estrogen(17β-estradiol)for bindingto ER.Methods 17β-estradiol-BSA conjugate is immobilized on themicroplates.In the second(competition)step the environmental sample isincubated together with a limited amount of ER.After the receptor bindingreaction,the supernatant,which contains environmental estrogen-ER complex,is incubated in another microplate coated with anti-ER antibody.Because ERhomodimerization can bind to specific sites on DNA- estrogen response elements(EREs),Nanoparticle -modified ERE probes are added and incubated.Finally the signals are amplified by silver enhancement and recorded withabsorbance.The intensity of signal is proportional to the quantity ofenvironmental estrogen in the sample.Results The assay result showed that aslittle as 100pg/L of 17β-estradiol could be detected and have a linear range from100pg/L to 1μg/L(R~2=0.9764).Conclusion The gold nanoparticle -modifiedERE assay is a screening method for the detection of environmental estrogenswith reliable,low cost,rapid,high-throughout and could be performed onmicroplates or chips.Compared with the existing methods,the detection limitcan be improved 2-3 orders of magnitude in this assay.
引文
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