17β-雌二醇对斑马鱼的毒性研究
发布时间：2019-01-10 19:46
【摘要】：环境中存在多种化合物都具有天然雌激素效应。这些化合物能干扰生物内分泌系统的正常功能,危害个体及其子代的健康,被统称为环境雌激素。其中以17β-雌二醇(17β-Estrodiol,E2)为其中最典型的代表。本研究选用斑马鱼作为实验材料,采用实时荧光定量反转录链式聚合酶反应(qRT-PCR)、转录组测序、蛋白质组iTraq定量分析等技术,探究了雌二醇对斑马鱼的毒性机制。以乙醇为助溶剂,配制0、10 ng/L、1、2、2.5、3、4、5、6 mg/L九个梯度浓度的17β-雌二醇溶液,对斑马鱼胚胎进行毒性处理,在不同的时间点(50 hpf、60 hpf、70 hpf、106 hpf、9 dpf)后,在显微镜下观察其对斑马鱼胚胎发育、形态变化和孵化率的影响。结果表明,经雌二醇溶液处理的胚胎会产生畸形,包括脊柱弯曲、心包水肿等,严重者直接死亡。此外,雌二醇还可以延迟斑马鱼胚胎的孵化。总之,经雌二醇处理后的胚胎的畸形率、死亡率和延迟孵化的程度均呈现一定的剂量效应。采用qRT-PCR定量分析了雌二醇对斑马鱼胚胎中孵化相关基因(zhe1)表达水平的影响,探究E2延迟斑马鱼孵化的分子机制。结果发现高浓度的E2能延迟zhe1的转录水平达到峰值所需的时间。之后,选用2 mg/L的雌二醇溶液对斑马鱼幼鱼(10 hpf)进行处理,选用10 ng/L的雌二醇溶液分别对斑马鱼幼鱼、21dpf的鱼、成年雌鱼(6月龄)、成年雄鱼(6月龄)进行处理,处理时长为9天。处理后,用qRT-PCR的方法分别检测各个处理组的斑马鱼体内性别分化相关基因(brca2,sox9a,sox9b,dmrt1和cyp19a1a)的表达水平的变化。结果表明:E2能上调雌性相关基因(brca2、sox9b)的表达,下调部分雄性相关基因(sox9a)的表达,对不同发育阶段的斑马鱼体内的性激素转化通路(cyp19a1a)的影响不同。选用0、10 ng/L的雌二醇溶液分别对斑马鱼幼鱼(10 hpf)进行处理。9天后,收集幼鱼,进行转录组测序和蛋白组的同位素标记法相对与绝对定量(iTRAQ)分析。转录组分析结果表明:经E2处理后,斑马鱼体内有82个基因上调和236个基因下调。经GO和KEGG注释和归纳分析后,得到雌二醇对斑马鱼的主要毒性影响:减缓新陈代谢(降低转录和翻译的效率,抑制血管形成,抑制增殖,抑制凋亡),抑制胆汁酸和甾类激素的合成,加快ATP的消耗(编码ATPase的基因上调表达,嘌呤代谢减缓),诱导斑马鱼启动自我保护机制(增强皮肤屏障作用)。经蛋白组iTRAQ分析后得到雌二醇对斑马鱼的主要毒性影响为:抑制细胞增殖和增强皮肤屏障作用,这与转录组学的结果部分一致。本研究从分子水平、转录组水平和蛋白组水平上,研究了雌二醇对不同发育阶段斑马鱼的毒性机制。为环境雌激素的毒性研究及其排放标准的制定提供了参考。
[Abstract]:Many compounds in the environment have natural estrogenic effects. These compounds interfere with the normal functions of the biological endocrine system and endanger the health of individuals and their offspring. Among them, 17 尾-estradiol (17 尾-Estrodiol,E2) is the most typical one. In this study, zebrafish were used as experimental materials, real-time fluorescence quantitative reverse transcription-chain polymerase reaction (qRT-PCR), transcriptome sequencing and proteome iTraq quantitative analysis were used to explore the toxic mechanism of estradiol on zebrafish. Using ethanol as cosolvent, a solution of 17 尾 -estradiol with 9 gradient concentrations of 0 ~ 10 ng/L,1,2,2.5,3,4,5,6 mg/L was prepared. Zebrafish embryos were treated with toxicity at different time points (50 hpf,60 hpf,70 hpf,106 hpf,). The effects of 9 dpf on the development, morphological change and hatching rate of zebrafish embryos were observed under microscope. The results showed that embryos treated with estradiol solution could produce deformities, including spinal curvature, pericardial edema, and death in severe cases. In addition, estradiol can delay the hatching of zebrafish embryos. In conclusion, the deformity rate, mortality and delayed hatching of embryos treated with estradiol showed a dose effect. The effects of estradiol on the expression of incubation-related genes (zhe1) in zebrafish embryos were quantitatively analyzed by qRT-PCR, and the molecular mechanism of E2 delayed incubation of zebrafish was explored. It was found that high E 2 concentration could delay the time required for zhe1 transcription to reach its peak. After that, juvenile zebrafish (10 hpf) were treated with estradiol solution for 2 mg/L, juvenile zebrafish, 21dpf fish and adult female (6 months old) were treated with estradiol solution for 10 ng/L, respectively. Adult male fish (6 months old) were treated for 9 days. After treatment, the expression levels of sex differentiation related genes (brca2,sox9a,sox9b,dmrt1 and cyp19a1a) in zebrafish were detected by qRT-PCR. The results showed that E2 could up-regulate the expression of female related genes (brca2,sox9b), down-regulate the expression of some male related genes (sox9a), and have different effects on the sex hormone transformation pathway (cyp19a1a) in zebrafish at different developmental stages. Juvenile zebrafish (10 hpf) were treated with estradiol solution for 10 ng/L. After 9 days, juvenile fish were collected, sequenced by transcriptome and analyzed by isotope labeling and absolute quantitative (iTRAQ). Transcriptome analysis showed that 82 genes were up-regulated and 236 genes down-regulated in zebrafish after E2 treatment. The main toxic effects of estradiol on zebrafish were summarized and analyzed by GO and KEGG. The main toxic effects of estradiol on zebrafish were as follows: slowing down metabolism (reducing the efficiency of transcription and translation, inhibiting angiogenesis, inhibiting proliferation, inhibiting apoptosis). Inhibit the synthesis of bile acids and steroid hormones, accelerate the consumption of ATP (up-regulation of ATPase gene expression, slow down of purine metabolism), and induce zebrafish to initiate self-protection mechanism (enhance skin barrier). The main toxic effects of estradiol on zebrafish were as follows: inhibition of cell proliferation and enhancement of skin barrier, which were consistent with the results of transcriptology. In this study, the toxicity of estradiol to zebrafish at different developmental stages was studied at molecular, transcriptional and proteome levels. It provides a reference for the study of the toxicity of environmental estrogens and the formulation of emission standards.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X171.5
本文编号：2406719
[Abstract]:Many compounds in the environment have natural estrogenic effects. These compounds interfere with the normal functions of the biological endocrine system and endanger the health of individuals and their offspring. Among them, 17 尾-estradiol (17 尾-Estrodiol,E2) is the most typical one. In this study, zebrafish were used as experimental materials, real-time fluorescence quantitative reverse transcription-chain polymerase reaction (qRT-PCR), transcriptome sequencing and proteome iTraq quantitative analysis were used to explore the toxic mechanism of estradiol on zebrafish. Using ethanol as cosolvent, a solution of 17 尾 -estradiol with 9 gradient concentrations of 0 ~ 10 ng/L,1,2,2.5,3,4,5,6 mg/L was prepared. Zebrafish embryos were treated with toxicity at different time points (50 hpf,60 hpf,70 hpf,106 hpf,). The effects of 9 dpf on the development, morphological change and hatching rate of zebrafish embryos were observed under microscope. The results showed that embryos treated with estradiol solution could produce deformities, including spinal curvature, pericardial edema, and death in severe cases. In addition, estradiol can delay the hatching of zebrafish embryos. In conclusion, the deformity rate, mortality and delayed hatching of embryos treated with estradiol showed a dose effect. The effects of estradiol on the expression of incubation-related genes (zhe1) in zebrafish embryos were quantitatively analyzed by qRT-PCR, and the molecular mechanism of E2 delayed incubation of zebrafish was explored. It was found that high E 2 concentration could delay the time required for zhe1 transcription to reach its peak. After that, juvenile zebrafish (10 hpf) were treated with estradiol solution for 2 mg/L, juvenile zebrafish, 21dpf fish and adult female (6 months old) were treated with estradiol solution for 10 ng/L, respectively. Adult male fish (6 months old) were treated for 9 days. After treatment, the expression levels of sex differentiation related genes (brca2,sox9a,sox9b,dmrt1 and cyp19a1a) in zebrafish were detected by qRT-PCR. The results showed that E2 could up-regulate the expression of female related genes (brca2,sox9b), down-regulate the expression of some male related genes (sox9a), and have different effects on the sex hormone transformation pathway (cyp19a1a) in zebrafish at different developmental stages. Juvenile zebrafish (10 hpf) were treated with estradiol solution for 10 ng/L. After 9 days, juvenile fish were collected, sequenced by transcriptome and analyzed by isotope labeling and absolute quantitative (iTRAQ). Transcriptome analysis showed that 82 genes were up-regulated and 236 genes down-regulated in zebrafish after E2 treatment. The main toxic effects of estradiol on zebrafish were summarized and analyzed by GO and KEGG. The main toxic effects of estradiol on zebrafish were as follows: slowing down metabolism (reducing the efficiency of transcription and translation, inhibiting angiogenesis, inhibiting proliferation, inhibiting apoptosis). Inhibit the synthesis of bile acids and steroid hormones, accelerate the consumption of ATP (up-regulation of ATPase gene expression, slow down of purine metabolism), and induce zebrafish to initiate self-protection mechanism (enhance skin barrier). The main toxic effects of estradiol on zebrafish were as follows: inhibition of cell proliferation and enhancement of skin barrier, which were consistent with the results of transcriptology. In this study, the toxicity of estradiol to zebrafish at different developmental stages was studied at molecular, transcriptional and proteome levels. It provides a reference for the study of the toxicity of environmental estrogens and the formulation of emission standards.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X171.5
【参考文献】
相关期刊论文 前1条
1 吴玉萍,熊茜,张广献,徐安龙;斑马鱼基因工程的研究进展[J];遗传学报;2004年10期
，本文编号：2406719
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