环境激素对鱼类生殖影响生物检测方法的建立
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摘要
当前,环境激素的生态效应已经引起人类的极大关注。由于环境激素种类多样,并且在环境中存在水平极低,为化学检测带来困难。因此,为了评价环境激素的污染状况及潜在危害,生物检测方法越来越受到人们的重视。在对环境激素的研究中,目前已经建立检测特异标志物——卵黄蛋白原的ELISA方法和普通RT-PCR方法。前者需要制备特异抗体,程序复杂,且相对于从分子水平进行的基因表达定量方法灵敏度仍有较大差距;而普通RT-PCR方法在基因表达定量方面也存在准确性较差的缺点,不能有效地反映剂量效应关系。本研究中通过对鱼类原代肝细胞暴露E2,采用Q-RT-PCR检测青鳉鱼和梭鱼VTG等基因的表达变化,期望建立较高灵敏度的环境雌激素效应评价方法。主要取得了如下研究结果:
(1)本实验通过对青鳉鱼肝脏细胞原代培养,建立了稳定的毒理学实验模型,与传统的活体暴露实验相比,节约了实验动物材料、实验周期及实验耗材。为环境激素毒理学研究奠定了基础。
(2)本研究中成功建立了实时定量RT-PCR与原代培养青鳉鱼肝细胞相结合的in vitro环境雌激素效应评价方法,并且显示出较高的灵敏度。
(3)VTG-I、VTG-II、CHG-H、CHG-L和ERα在体外培养青鳉鱼肝细胞中均与雌激素暴露浓度呈现较好的剂量效应关系,其中VTG-I、VTG-II、CHG-H、CHG-L作为检测雌激素的生物标记物基因可优先选择。
(4)本文通过扩增梭鱼VTG、β-actin基因,测定基因序列,建立检测E2暴露后梭鱼肝细胞VTG表达的实时定量RT-PCR方法。为环境激素的动态检测和分析鱼类对内分泌干扰物质的敏感性分析提供了方法学基础。
More and more attentions have been focused on the issue of endocrine disrupting chemicals (EDCs) in the environment in the worldwide, recently. It is challenging to study EDCs in the environment for instrumental methods because of various low level EDCs. To assess the potential impacts, biomarkers become effective method for analyzing the effects of EDCs in the environment. Of these biomarkers, vitellogenin (VTG) has been used as an important biomarker for monitoring EDCs, and easiest and most automated assay are designed around ELISA or general RT-PCR to detect VTG in fishes. The former needs of specific antibodies, complicated procedures, and compared to the molecular level of gene expression quantitative methods are still a wide gap between sensitivity;The general RT-PCR method in quantitative gene expression There is also the shortcomings of poor accuracy, can not effectively reflect the dose-effect relationship. In this study by medaka fish primary hepatocytes exposed E2, using Q-RT-PCR detection, such as fish and Barracuda VTG gene expression changes, the establishment of higher expectations of the environmental sensitivity of estrogen effect of evaluation methods. The following achievements are obtained:
(1) This study by medaka primary hepatocytes cultured in order to establish stability in the toxicology test model, with the traditional live exposure experiment compared with the experimental animal conservation materials, supplies and laboratory experiments cycle. For endocrine disrupting chemicals lay the foundation for toxicology studies.
(2) This study successfully established a real-time quantitative RT-PCR and the culture of medaka primary hepatocytes in vitro combination of environmental effects of estrogen evaluation methods, and showed high sensitivity.
(3) VTG-I, VTG-II, CHG-H, CHG-L and ERαin vitro medaka primary hepatocytes were exposed with estrogen showed good dose-response relationship, which VTG-I, VTG-II, CHG - H, CHG-L as an estrogen detection of biological markers of the first option.
(4) In this paper, amplified so-iuy mullet VTG,β-actin genes, gene sequence determination, the establishment of E2 detection of liver cells exposed so-iuy mullet VTG expression of real-time quantitative RT-PCR method. The dynamic environment for hormone testing and analysis of fish on endocrine disruptors the sensitivity analysis provides a methodology based.
(1)本实验通过对青鳉鱼肝脏细胞原代培养,建立了稳定的毒理学实验模型,与传统的活体暴露实验相比,节约了实验动物材料、实验周期及实验耗材。为环境激素毒理学研究奠定了基础。
(2)本研究中成功建立了实时定量RT-PCR与原代培养青鳉鱼肝细胞相结合的in vitro环境雌激素效应评价方法,并且显示出较高的灵敏度。
(3)VTG-I、VTG-II、CHG-H、CHG-L和ERα在体外培养青鳉鱼肝细胞中均与雌激素暴露浓度呈现较好的剂量效应关系,其中VTG-I、VTG-II、CHG-H、CHG-L作为检测雌激素的生物标记物基因可优先选择。
(4)本文通过扩增梭鱼VTG、β-actin基因,测定基因序列,建立检测E2暴露后梭鱼肝细胞VTG表达的实时定量RT-PCR方法。为环境激素的动态检测和分析鱼类对内分泌干扰物质的敏感性分析提供了方法学基础。
More and more attentions have been focused on the issue of endocrine disrupting chemicals (EDCs) in the environment in the worldwide, recently. It is challenging to study EDCs in the environment for instrumental methods because of various low level EDCs. To assess the potential impacts, biomarkers become effective method for analyzing the effects of EDCs in the environment. Of these biomarkers, vitellogenin (VTG) has been used as an important biomarker for monitoring EDCs, and easiest and most automated assay are designed around ELISA or general RT-PCR to detect VTG in fishes. The former needs of specific antibodies, complicated procedures, and compared to the molecular level of gene expression quantitative methods are still a wide gap between sensitivity;The general RT-PCR method in quantitative gene expression There is also the shortcomings of poor accuracy, can not effectively reflect the dose-effect relationship. In this study by medaka fish primary hepatocytes exposed E2, using Q-RT-PCR detection, such as fish and Barracuda VTG gene expression changes, the establishment of higher expectations of the environmental sensitivity of estrogen effect of evaluation methods. The following achievements are obtained:
(1) This study by medaka primary hepatocytes cultured in order to establish stability in the toxicology test model, with the traditional live exposure experiment compared with the experimental animal conservation materials, supplies and laboratory experiments cycle. For endocrine disrupting chemicals lay the foundation for toxicology studies.
(2) This study successfully established a real-time quantitative RT-PCR and the culture of medaka primary hepatocytes in vitro combination of environmental effects of estrogen evaluation methods, and showed high sensitivity.
(3) VTG-I, VTG-II, CHG-H, CHG-L and ERαin vitro medaka primary hepatocytes were exposed with estrogen showed good dose-response relationship, which VTG-I, VTG-II, CHG - H, CHG-L as an estrogen detection of biological markers of the first option.
(4) In this paper, amplified so-iuy mullet VTG,β-actin genes, gene sequence determination, the establishment of E2 detection of liver cells exposed so-iuy mullet VTG expression of real-time quantitative RT-PCR method. The dynamic environment for hormone testing and analysis of fish on endocrine disruptors the sensitivity analysis provides a methodology based.
引文
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[2] Richard S. Environmental estrogens stir debate[J]. Science, 1994, 265 (15): 308- 310.
[3] Wright AN. Natural and anthropogenic environmental estrogens: The Scientific Basis for Risk Assessment[J]. Pure and Applied Chemistry, 1998, 70 (9): 1713- 1723.
[4] Crews D, Willingham E, Skipper JK. Endocrine disruptors: Present issues, future directions[J]. Quarterly Review of Biology, 2000, 75 (3): 243- 260.
[5] Allen Y, Smith A, Scott, AP, Thain JE, Kirby MF and Matthiessen P. Endocrine disruption in the marine environment (EDMAR)[R]. Final Report, 2002.
[6] Warren G, Foster. Endocrine Disruption and Human Reproductive Effects: An Overview[J]. Water Quality Research Journal of Canada, 2001, 36 (2): 253- 271.
[7] Bounias M. Etiological factors and mechanism involved in relationships between pesticide exposure and cancer[J]. Journal of Environmental Biology, 2003, 24 (1): 1- 8.
[8] Swan SH, Elkin EP, Fenster L. Have sperm densities declined? A reanalysis of global trend data[J]. Environmental Health Perspectives. 1997, 105: 1228- 1232.
[9] Carlsen E, Giwercman A, Keiding N, SkakkebFk NE. Evidence for decreasing quality of semen during past 50 years[J]. British Medical Journal, 1992, 305: 609- 613.
[10] Swan SH, Elkin EP, Fenster L. Have sperm densities declined? A reanalysis of global trend data[J]. Environmental Health Perspectives, 1997, 105: 1228- 1232.
[11] Blackmore G. Imposex in Thais clavigera (neogastropoda) as an indicator of TBT (tributyltin) bioavailability in coastal waters of Hong Kong[J]. Journal of Molluscan Studies, 2000, 66: 1- 8.
[12] Jobling S, Tyler CR. Endocrine disruption in wild freshwater fish[J]. Pure and Applied Chemistry, 2003, 75 (11- 12): 2219- 2234.
[13] De Metrio G, Corriero A, Desantis S, Zubani D, Cirillo F, Deflorio M, Bridges CR, Eicker J, de la Serna J M, Megalofonou P, Kime DE. Evidence of a high percentage of intersex in the Mediterranean swordfish (Xiphias gladius L.)[J]. Marine Pollution Bulletin, 2003, 46 (3): 358- 361.
[14] Jobling S, Nolan M, Tyler CR, Brighty G, Sumpter JP. Widespread sexual disruption in wild fish[J]. Environmental Science & Technology, 1998, 32 (17): 2498- 2506.
[15] Piferrer F. Endocrine sex control strategies for the feminization of teleost fish[J]. Aquaculture, 2001, 197: 229- 281.
[16] Fossi MC, Silvia C, Stefania A, Alessandra M, Antonella A, Giuseppe N S. Do endocrine disrupting chemicals threaten Mediterranean swordfish? Preliminary results of vitellogenin and Zona radiata proteins in Xiphias gladius[J]. Marine Environmental Research, 2001, 52: 477- 483.
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