直观检测环境雌激素的转基因斑马鱼系的建立及其应用研究

【作者】 陈浩；

【导师】 宋后燕； 汤其群； 马端； 于敏；

【作者基本信息】 复旦大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 环境雌激素（EEs）是指能够模拟体内天然雌激素功能的人工合成或植物提取的化合物。这些物质已被证明能够导致动物和人类的性别混淆和生殖功能受损。此外,它们还能诱导雄性和幼年鱼类表达雌性特异性蛋白,如卵黄蛋白原（VTG）。为了能快速、直观、活体检测环境雌激素,我们建立了转基因斑马鱼系。首先,我们用RT-PCR和整体原位杂交的方法显示了雌激素类化合物17α-炔雌醇（EE₂）诱导斑马鱼卵黄蛋白原1（zvtgl）基因在胚胎和幼鱼体内的表达模式。第二,我们克隆了zvtgl基因上游两段调控序列,并使其在MCF-7细胞株中诱导表达下游绿色荧光蛋白基因（gpf）,由此体外验证1720bp DNA片段具有启动子活性。第三,融合基因zvtgl:gfpl显微注射1-2细胞期的斑马鱼胚胎中,经EE₂处理后幼鱼肝脏出现绿色荧光。由此从活体验证1720bp DNA片段的启动子活性。第四,为了增强转基因斑马鱼绿色荧光蛋白的表达,我们合成了一段来源于爪蟾卵黄蛋白原A2基因的39bp的DNA片段,该片段含有雌激素反应元件（ere）的序列。这段DNA插入zvtgl启动子的上游,得到融合基因ere-zvtgl:gfp,并显微注射斑马鱼胚胎。然后用10ng/L的EE₂处理6天,用荧光显微镜观察肝脏荧光,进行筛选。出现荧光的幼鱼继续饲养至性成熟,与野生型斑马鱼杂交,其后代也用10ng/L的EE₂处理6天,观察荧光进行筛选。在119条F0代斑马鱼中,发现一条能将荧光传递到F1代。最后只有两条F1代雄鱼存活下来,并和野生型雌鱼杂交得到F2代。F2代的雌鱼和雄鱼互相交配,得到F3代。F3再和野生型杂交,用EE₂筛选出现荧光的F4代。最后,能将荧光100%传递到F4代的F3代被确定为纯合子,从而建立ere-zvtgl:gfp转基因斑马鱼系。在这种转基因斑马鱼系中,绿色荧光蛋白（GFP）特异性地在成熟雌鱼肝脏表达。雄性和幼年转基因斑马鱼不表达GFP,但是在EE₂诱导后其肝脏也能出现荧光。胚胎和幼鱼中zvtgl mRNA和绿色荧光出现的时空一致,说明gfp基因是存zvtgl启动子的调控下表达。1-2细胞期的转基因斑马鱼分别用100,10,1,0.1 ng/L的EE₂处理,最早观察到荧光的时间分别为53,74,100和131小时。成年雄性转基因斑马鱼分别用100,10,1,0.1 ng/L的EE₂处理,最早观察到荧光的时间分别为2,3,4,和7天。成熟雌性转基因斑马鱼虽然肝脏已经有荧光,但是相对较高浓度（10和100ng/L）的EE₂仍然能够诱导荧光增强。随着暴露时间的延长,肝脏的绿色荧光有增强趋势,而撤去EE₂后一段时间荧光逐渐消失。分别用具有雌激素活性的化合物和没有雌激素活性的甾体类激素测试ere-zvtgl:gfp转基因斑马鱼发射荧光的反应,结果雌激素类物质处理幼鱼13天后,诱导荧光所需的浓度分别为:已烯雌酚50 ng/L,雌二醇0.1μg/L,雌三醇1μg/L,氯化镉10μg/L,玉米赤霉烯酮50μg/L,双酚A 1mg/L。弱雌激素壬基酚浓度达到10mg/L时仍未能诱导绿色荧光出现。其它甾体类激素如孕激素和17-羟固醇即使浓度达到100mg/L和50mg/L,仍不能诱导荧光出现。这种转基因斑马鱼能检测浓度很低的环境雌激素,而且所需的暴露时间很短,因此有望应用于直观,快速,简便地监测环境雌激素。利用这种转基因斑马鱼系,研究EE₂诱导zvtgl表达的分子机制,结果提示雌激素受体β是EE₂诱导zvtgl表达所必须的;环境雌激素EE₂浓度达500ng/L时斑马鱼胚胎原始生殖细胞（PGCs）迁移异常,这种效应与雌激素受体无关。PGCs迁移异常对斑马鱼胚胎发育的影响正在研究中。更多还原

【Abstract】 Environmental estrogens（EEs） are synthetic chemical compounds and plant-derived substance that are capable of imitating the functions of natural in vivo estrogen hormones.It has been known that estrogenic endocrine disrupters,which can cause intersexuality and reproduction decline in animals and human,can also induce synthesis of female-specific proteins such as vitellogenin（VTG） in male and juvenile fish.To establish a novel in vivo test system for rapidly and visibly detection of environmental estrogens,a transgenic zebrafish line was developed.First,the expression pattern of zvtg1 was demonstrated by RT-PCR and whole mount in situ hybridization in developing zebrafish exposed to synthetic estrogen 17αethinylestradiol（EE₂）.Second,two DNA fragments upstream zvtg1 gene was cloned and the 1720bp fragment was identified as effective promoter in vitro by driving reporter green fluorescent protein（gfp） gene expression in MCF-7 cell line.Third,the zvtg1:gfp fusion DNA was microinjected into 1～2-cell stage embryos and faint green fluorescence in the liver was observed in larvae exposed to EE₂.Thus the promoter activity of 1720bp DNA fragment was identified in vivo.Fourth,in order to increasing GFP expression in transgenic zebrafish,a 39 base pairs DNA sequences contained the consensus estrogen response element（ere,GGTCAnnnTGACC） was synthesized according to the 5’end region of Xenopus vitellogenin A₂ gene,and was added upstream of the zvtg1 promoter.Then the fusion gene ere-zvtg1:gfp was microinjected into 1-2 cell stage embryos.Injected embryos were treated with 10ng/L EE₂ for 6 days and screened by examining fluorescence in the liver by using a fluorescence microscope.Founders were raised till sexually mature and individually mated with wild-type fish,and the offspring were examined fluorescence after being treated with 10ng/L EE₂ for 6 days.Of the 119 founders one germline-transmitted transgenic fish was identified.F1 fish fry obtained from the positive founder were screened by observing fluorescence and the positive F1 fish were raised.Two positive male F1 fish were survived and mated with wide type females to generate F2 fish.F2 males were mated with F2 females to breed F3 generation which was intercrossed with wide type zebrafish to generate F4 fish.Finally,the ere-zvtg1:gfp transgenic line was confirmed by 100%fluorescence induction in the F4 generation.In this transgenic line,GFP was exclusively expressed in the liver of the mature adult female.Male and larval transgenic fish did not express GFP but could be induced to express GFP in the liver after exposure to 17α-ethynylestradiol（EE₂）. Concurrent of zvtg1 and gfp expression in embryos and larvae after EE₂ exposure was observed,which indicated that the expression of gfp transgene was driven by the zvtg1 promoter.Green fluorescence was first observed in the liver at 53,74,100 or 131 hours post fertilization（hpf） after exposure to 100,10,1 or 0.1 ng/L EE₂ respectively from 1-2 cell stage.As for mature male transgenic zebrafish,green fluorescence was observed after exposure to 100,10,1 or 0.1 ng/L EE₂ for 2,3,4 or 7 days respectively; as for mature female,fluorescence enhanced after exposure to relatively high concentrations of EE₂（10 and 100 ng/L）.Green fluorescence in the liver was increased with prolonging of exposure time and was repeatedly induced after removal and re-addition of EE₂.We also demonstrated that GFP expression could be induced by other estrogenic compounds,includingβ-estradiol（E₂,0.1μg/L）,Cadmium chloride（CdCl₂,10μg/L）,zearalenone（50μg/L）,estriol（E₃,1μg/L ）, diethylstilbesterol（DES,50 ng/L） bisphenol A（BPA,1 mg/L）but not by week estrogenic compounds such as nonylphenol（NP,up to 10 mg/L）,or non-estrogenic steroid hormone such as progesterone（up to 100 mg/L） and 17hydroxysteroid（up to 50 mg/L）.These data suggest the transgenic zebrafish is sensitive and specific for detection of estrogenic compounds.Because the observed-effective concentrations are as low as those of environment and the observed-effective exposure times are very short,this transgenic fish is hopeful for monitoring environmental estrogens directly. rapidly and easily.Using this transgenic line,we investigated the molecular mechanism involved in EE₂ caused feminination and abnormal gonads in zebrafish.The results suggested estrogen receptorβwas needed for EE₂ induced zvtg1 expression in larval zebrafish. We also demonstrated high concentration of EE₂（500ng/L） disturbed the primordial germ cells（PGCs） migration in embryonic zebrafish,and this effect seemed having no relationship with estrogen receptors.更多还原