【作者】 刘宝敏；

【导师】 袁东星；

【作者基本信息】 厦门大学 ， 环境科学， 2008， 博士

【摘要】 卵黄蛋白原（Vitellogenin,Vtg）是卵生动物中卵黄蛋白的前体,通常只存在于性成熟的雌性个体中。但是如果雄性或幼体暴露于环境雌激素环境,其体内也能产生Vtg。因此,Vtg被认为是一种理想的环境雌激素蛋白质生物标志物,在研究各种化合物的雌激素效应中被广泛应用。Vtg的纯化多采用离子交换与凝胶渗透两步骤法,耗时长,回收率低、Vtg容易降解。因此,有必要开发一种简单、快速、回收率高的Vtg纯化方法。目前Vtg的检测主要采用特异性的免疫分析方法,方法灵敏,选择性强;但耗时长,操作繁琐。建立快速、简便、可靠的Vtg检测方法成为深入开展Vtg作为环境雌激素标志物研究的关键前提。在环境雌激素毒性研究方面,大多数研究仅针对单一环境雌激素的毒性效应展开,而对多种雌激素的复合污染和联合效应的研究较少。随着我国沿海地区经济的不断发展,大量污染物被排入大海,沿海及河口地区的污染日益加剧,开展持久性强、毒性大的环境雌激素对近海海洋生物联合效应的研究,因而具有重要意义。本研究将离子交换色谱和超滤离心技术相结合,建立了两级纯化Vtg的新方法;将内源荧光法与紫外分光法相结合,建立了灵敏度高、线性范围广、操作简单的Vtg测定新方法;并在建立分离检测手段的基础上,利用Vtg为主要观察指标,开展己烯雌酚（DES）和炔雌醇（EE₂）联合作用研究。本研究的主要内容和结果包括以下几个方面:1.卵黄蛋白原的两级纯化与初步鉴定采用17β-雌二醇（E₂）腹腔注射的方式诱导真鲷（Pagrosomus major）产生大量Vtg,利用弱阴离子交换和超滤两级纯化的方法,快速、可靠地获得了纯度和浓度较高、盐度较低的Vtg实验室标样。整个纯化过程省时简便。两级纯化得到的Vtg的色谱、电泳行为及分子量与文献报道的基本一致。2.内源荧光法与紫外分光法串联测定鱼血清中的Vtg利用Mini Q高分辨离子交换色谱柱对鱼血清样品进行分离,采用紫外分光法和内源荧光法同时测定Vtg,建立了一种线性范围宽、灵敏度高、操作简单的测定方法。紫外分光法测定Vtg的线性范围为24.1-96.4μg/mL,检出限为8μg/mL（S/N=5）;荧光法测定Vtg的线性范围为1.9-30μg/mL,检出限为1μg/mL（S/N=5）。分别对鱼血清样品进行5次平行测定,紫外分光法与内源荧光法检测的相对标准偏差（RSD）分别为2.4%（n=5）及3.4%（n=5）。该方法操作简单、快速、重现性好。将本方法应用于美国红鱼（Sciaenops ocellatus）血清中Vtg的测定,结果发现美国红鱼血清中Vtg的离子交换色谱行为与真鲷鱼血清Vtg的相似,说明本方法通用性较强。3.Vtg作为环境雌激素生物标志物的研究以Vtg作为生物标志物,研究了DES、EE₂及两者的混合物对美国红鱼幼鱼的浓度-效应及时间-效应关系,并以Vtg为主要指标考察了DES和EE₂对美国红鱼的联合雌激素效应。结果如下:（1）DES和EE₂对美国红鱼幼鱼具有一定的毒性作用,可使个体异常,严重者死亡。（2）DES、EE₂及两者的混合物均使美国红鱼肥满度降低、肝胰脏指数与肾脏指数增加。雌激素浓度增加及暴露时间延长,均使这种影响的结果加重。浓度梯度实验中,DES、EE₂及两者的混合物对肝脏指数的影响最明显,对肥满度的影响最小;时间序列实验中,雌激素对肥满度的影响在第30 d显著性出现,对肾脏指数的影响在第18 d显著性出现,而对肝胰脏指数的影响则在第9 d就显著性出现。（3）DES、EE₂及两者的混合物在较低浓度（三者浓度分别小于320、160和120ng/L）时,随浓度的增加美国红鱼血清总蛋白浓度及Vtg浓度增加。但随着雌激素浓度的进一步提高,血清总蛋白及Vtg的浓度逐渐降低,出现典型的Hormesis现象。随着暴露时间的增加,血清总蛋白及Vtg的浓度逐渐增加,体现了很好的时间-效应关系。与各项生理指标及血清总蛋白浓度相比,Vtg浓度的变化最显著、最迅速,最能体现DES和EE₂的雌激素效应,说明Vtg是一种较好的环境雌激素生物标志物。（4）在Vtg浓度与各指标之间的关系中,Vtg与肥满度没有相关性,而Vtg浓度与血清总蛋白有很好的正相关性（P＜0.001）。Vtg浓度与肝胰脏指数和肾脏指数的相关性有一定的相似性:即Vtg浓度与二者之间有一定的相关性（P＜0.05）;当Vtg浓度较低时,这种相关性更加明显（P值小于或接近0.01）;而当Vtg浓度高时,与两者的相关性降低（P＞0.05）。（5）DES和EE₂对美国红鱼幼鱼的剂量-效应关系符合典型的S形剂量-效应关系,用Logistic模型能很好地拟合。采用浓度相加模式和独立作用模式,可预测DES与EE₂的联合效应。与独立作用模式相对比,浓度相加模式能更好地预测DES与EE₂的联合雌激素效应。更多还原

【Abstract】 Vitellogenin（Vtg） is the egg yolk protein precursor in the oviparous animals.It is usually produced in sexually active female individuals.However,Vtg can also be detected in the male and the juvenile exposed to estrogen or estrogen mimic chemicals.Therefore,Vtg in male serum or plasma is generally considered to be a candidate of protein biomarker for environmental estrogens,thus widely used for the study of estrogenic effect of chemicals.Currently,most approaches for Vtg purification are the combination method of ion exchange chromatography and gel permeation chromatography,which suffer from low recovery,time-consuming and easy degradation for protein.Therefore,it is necessary to develop a new purification protocol that is simple,rapid and has high recovery.Specific assay methods termed immunoassays have been widely used for analysis of Vtg.It appears that immunological techniques applied to Vtg proteins have a great potential for developing into specific,sensitive quantitative methods.However, these techniques are cumbersome and require a long time for sample preparation.Therefore,the prerequisite for the study of environmental estrogens is to establish a rapid,reliable and valid method for the determination of Vtg in some species.Conventional ecotoxicity study of chemicals for their estrogenic effects has predominantly employed systems designed mostly for single estrogen,but few for the combinations.With the economical development along the coastal areas in China,a lot of discarded chemicals deteriorate the water quality of the coastal and estuary region.It is essential to investigate the joint effects of the persistent and toxic environmental estrogens on marine organisms.In this study,a two-step method had been developed for purification of Vtg with ultrafiltration and weak anion exchange liquid chromatography.And Vtg was determinated by the method of intrinsic fluorescence or UV.The method was sensitive, simple and with a wide linear range.Based on the established methods of the purification and the determination,the joint toxic effect of diethylstilbestrol（DES） and 17α-ethynylestradiol（EE₂） was studied using Vtg as the main monitoring index.The detail information,divided into three sections,is as follows.1.Two-step purification and primary characterization of VtgVtg from sea bream（Pagrosomus major） was induced by 17β-estradiol（E₂） with the application of intraperitoneal injection.With weak anion exchange liquid chromatography and ultrafiltration,a two-step method had been developed for rapid and reliable purification of Vtg from the serum of sea bream.It was found that most impurities and salt could be removed during the first step by weak anion exchange liquid chromatography and further clean-up in the second step through ultrafiltration. The concentrated Vtg purified with the two-step method had the same characteristics of chromatography,PAGE electrophoresis and protein molecular as those reported by other researchers.The purified Vtg could be used as the standard sample inner laboratory.The results showed that the two-step purification was simple and effective.2.Determination of Vtg in fish serum by HPLC with UV and intrinsic fluorescence detection in tandemA sensitive and simple determination method for Vtg in fish serum with a wide linear range was developed.The Vtg separated by high-resolution anion exchange chromatography was detected by intrinsic fluorescence and UV detection in tandem. The linearity curve ranged from 24.1 to 96.4μg/mL with a detection limit 8.0μg/mL （S/N=5） for UV detection and from 1.9 to 30μ/mL with a detection limit 1.0μg/mL （S/N=5） for fluorescence detection.The relative standard deviations（RSD） for UV detection and for fluorescence detection were 2.4%（n=5） and 3.4%（n=5）, respectively.The method was simple,rapid and replicable.The proposed method was successfully applied to determine Vtg in fish serum from red drum（Sciaenops ocellatus）.The validation results showed that the Vtg of red drum and sea bream had the same chromatographic behaviors.It indicated that the developed method could be applied to evaluate Vtg in various fishes. 3.Study of Vtg as a biomarker for environmental estrogensThe concentration-effect and exposed time-effect of DES,EE₂ and their mixture on red drum were studied.Moreover,with Vtg as the main biomarker,the joint estrogen effect of DES and EE₂ on red drum was investigated.The results are as follows.（1） DES and EE₂ were toxic to juvenile red drum.They could lead the individuals to abnormality,and even death.（2） The condition factor（CF）,hepatopancreas somatic index（HSI） and renal somatic index（RSI） increased with the increasing expose time and concentrations of DES,EE₂ and their mixture.The effect of DES,EE₂ and their mixture in the experiment of concentration series was most significant for HIS and lest for CF.The significant effect in the experiment ot:time series was found at the exposed time of 30 days for CF,18 days for RSI and 9 days for HIS,respectively.（3） The concentrations of total protein and Vtg in serum enhanced with the increasing concentrations of DES,EE₂ and their mixture at relatively low concentrations（less than 320,160 and 120 ng/L for DES,EE₂ and their mixture,respectively）.However,the concentrations of total protein and Vtg reduced with further increase of the concentrations.It demonstrated that the effect of DES,EE₂ and their mixture on total protein and Vtg accorded with Hormesis effect.The concentrations of total protein and Vtg were elevated with the prolonging expose time in the experiment of time series.Compared to other physiological indicators and the total serum protein,Vtg was the most significant and rapid one.It could well reflect the estrogenic effect of DES and EE₂.It was thus known that Vtg was a good candidate of protein biomarker for environmental estrogen.（4） From the relationships between Vtg and the other indicators,it was found that there was no significant correlation between Vtg and CF,but positive highly significant correlation（P＜0.001） between Vtg and total serum protein.The relationship between Vtg and HIS was similar to that between Vtg and RSI. There were significant correlations（P＜0.05） between Vtg and either HSI or RSI when the concentration of Vtg was in the range of 0-90 mg/mL.And there were highly significant correlations（P is less than or close to 0.01） between Vtg and the two when the concentration of Vtg was in the range of 0-25 mg/mL.However,there were no correlations（P＞0.05） between Vtg and the two when the concentration of Vtg was in the range of 25-90 mg/mL.（5） The relationships of concentration-effect for juvenile red drum exposed to DES and EE₂ coincided with the curve of typical s-shape.It could be fitted with the Logistic equation.Moreover,using the models of concentration addition and independent action it had shown that the joint effect of DES and EE₂ could be predicted from the activity of the individual chemicals. Compared to the model of independent action,the joint effect of the binary mixture could be well predicted by the model of concentration addition.更多还原