17β-雌二醇對(duì)斑馬魚(yú)的毒性研究
發(fā)布時(shí)間：2019-01-10 19:46
【摘要】：環(huán)境中存在多種化合物都具有天然雌激素效應(yīng)。這些化合物能干擾生物內(nèi)分泌系統(tǒng)的正常功能,危害個(gè)體及其子代的健康,被統(tǒng)稱(chēng)為環(huán)境雌激素。其中以17β-雌二醇(17β-Estrodiol,E2)為其中最典型的代表。本研究選用斑馬魚(yú)作為實(shí)驗(yàn)材料,采用實(shí)時(shí)熒光定量反轉(zhuǎn)錄鏈?zhǔn)骄酆厦阜磻?yīng)(qRT-PCR)、轉(zhuǎn)錄組測(cè)序、蛋白質(zhì)組iTraq定量分析等技術(shù),探究了雌二醇對(duì)斑馬魚(yú)的毒性機(jī)制。以乙醇為助溶劑,配制0、10 ng/L、1、2、2.5、3、4、5、6 mg/L九個(gè)梯度濃度的17β-雌二醇溶液,對(duì)斑馬魚(yú)胚胎進(jìn)行毒性處理,在不同的時(shí)間點(diǎn)(50 hpf、60 hpf、70 hpf、106 hpf、9 dpf)后,在顯微鏡下觀察其對(duì)斑馬魚(yú)胚胎發(fā)育、形態(tài)變化和孵化率的影響。結(jié)果表明,經(jīng)雌二醇溶液處理的胚胎會(huì)產(chǎn)生畸形,包括脊柱彎曲、心包水腫等,嚴(yán)重者直接死亡。此外,雌二醇還可以延遲斑馬魚(yú)胚胎的孵化�？傊�,經(jīng)雌二醇處理后的胚胎的畸形率、死亡率和延遲孵化的程度均呈現(xiàn)一定的劑量效應(yīng)。采用qRT-PCR定量分析了雌二醇對(duì)斑馬魚(yú)胚胎中孵化相關(guān)基因(zhe1)表達(dá)水平的影響,探究E2延遲斑馬魚(yú)孵化的分子機(jī)制。結(jié)果發(fā)現(xiàn)高濃度的E2能延遲zhe1的轉(zhuǎn)錄水平達(dá)到峰值所需的時(shí)間。之后,選用2 mg/L的雌二醇溶液對(duì)斑馬魚(yú)幼魚(yú)(10 hpf)進(jìn)行處理,選用10 ng/L的雌二醇溶液分別對(duì)斑馬魚(yú)幼魚(yú)、21dpf的魚(yú)、成年雌魚(yú)(6月齡)、成年雄魚(yú)(6月齡)進(jìn)行處理,處理時(shí)長(zhǎng)為9天。處理后,用qRT-PCR的方法分別檢測(cè)各個(gè)處理組的斑馬魚(yú)體內(nèi)性別分化相關(guān)基因(brca2,sox9a,sox9b,dmrt1和cyp19a1a)的表達(dá)水平的變化。結(jié)果表明:E2能上調(diào)雌性相關(guān)基因(brca2、sox9b)的表達(dá),下調(diào)部分雄性相關(guān)基因(sox9a)的表達(dá),對(duì)不同發(fā)育階段的斑馬魚(yú)體內(nèi)的性激素轉(zhuǎn)化通路(cyp19a1a)的影響不同。選用0、10 ng/L的雌二醇溶液分別對(duì)斑馬魚(yú)幼魚(yú)(10 hpf)進(jìn)行處理。9天后,收集幼魚(yú),進(jìn)行轉(zhuǎn)錄組測(cè)序和蛋白組的同位素標(biāo)記法相對(duì)與絕對(duì)定量(iTRAQ)分析。轉(zhuǎn)錄組分析結(jié)果表明:經(jīng)E2處理后,斑馬魚(yú)體內(nèi)有82個(gè)基因上調(diào)和236個(gè)基因下調(diào)。經(jīng)GO和KEGG注釋和歸納分析后,得到雌二醇對(duì)斑馬魚(yú)的主要毒性影響:減緩新陳代謝(降低轉(zhuǎn)錄和翻譯的效率,抑制血管形成,抑制增殖,抑制凋亡),抑制膽汁酸和甾類(lèi)激素的合成,加快ATP的消耗(編碼ATPase的基因上調(diào)表達(dá),嘌呤代謝減緩),誘導(dǎo)斑馬魚(yú)啟動(dòng)自我保護(hù)機(jī)制(增強(qiáng)皮膚屏障作用)。經(jīng)蛋白組iTRAQ分析后得到雌二醇對(duì)斑馬魚(yú)的主要毒性影響為:抑制細(xì)胞增殖和增強(qiáng)皮膚屏障作用,這與轉(zhuǎn)錄組學(xué)的結(jié)果部分一致。本研究從分子水平、轉(zhuǎn)錄組水平和蛋白組水平上,研究了雌二醇對(duì)不同發(fā)育階段斑馬魚(yú)的毒性機(jī)制。為環(huán)境雌激素的毒性研究及其排放標(biāo)準(zhǔn)的制定提供了參考。
[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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：X171.5
本文編號(hào)：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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：X171.5
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 吳玉萍,熊茜,張廣獻(xiàn),徐安龍;斑馬魚(yú)基因工程的研究進(jìn)展[J];遺傳學(xué)報(bào);2004年10期
，本文編號(hào)：2406719
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