以青魚將魚之初代肝臟培養細胞探討廢水對其卵黃先質(VTG)表現之影響

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標題:	以青魚將魚之初代肝臟培養細胞探討廢水對其卵黃先質(VTG)表現之影響 Using Japanese Medaka Primary Hepatic Cell Culture to Investigate the Effect of Wastewater on Expression of VTG
作者:	蘇于菁 Yu-Ching Su
貢獻者:	陳健民嘉南藥理科技大學：環境工程與科學系碩士班
關鍵字:	肝臟初代細胞離體試驗卵黃先質蛋白環境荷爾蒙 environmental hormones in vitro vitellogenesis primary hepatocyte cultures
日期:	2005
上傳時間:	2008-10-31 16:15:04 (UTC+8)
摘要:	近十年來，釵h學者熱烈的討論環境荷爾蒙相關議題，有些物質會模仿生物體內正常荷爾蒙的作用，或抑制其正常的作用，改變生物體內免疫、神經和內分泌系統的正常運作，這類化學物質統稱為環境荷爾蒙(environmental hormone)或內分泌干擾物(endrcrine disrupting substance, EDCs)，仿激素種類相當多而使用也廣泛，因此在一般水體中或污水處理廠排放水中皆可發現這類仿激素的存在。本研究主要在探討，都市廢水處理廠(sewage-treatment works，STWs)及人工溼地(westland)生活污水這兩類污水對於魚體卵黃先質蛋白(vitellogen，VTG)的誘導影響。因水生毒物研究須利用活體生物進行研究，而必須犧牲釵h個體方可檢測岀對生物的影響，本研究將初步建立青魚將魚(Oryzias latipes)肝臟初代細胞，並檢測魚體肝臟細胞中卵黃先質蛋白VTG含量及探討其做為受仿雌激素的誘導的可行性研究。將青魚將魚肝臟初代細胞暴露在含有培養基中(DMSO含量0.1％)，每天更換培養基，經過5、10及15天的暴露，最後將細胞以西方點墨法分析VTG的含量，最後再與相對參考毒物17-βEstradiol比較其相對量。結果發現，參考毒物在經暴露15天後細胞其暴露劑量與VTG產生量成劑量反應關係。在暴露組中，人工濕地大部分的進流水皆必須在第15天才可檢測到VTG的誘導反應，而放流水中僅安平都市污水有誘發VTG反應，且有反應的樣本其暴露時間與誘發反應成正比。因此，利用青魚將魚肝臟初代細胞檢測生活污水中仿雌激素誘導魚體內VTG是可行的。 In the last decade, much of savant efforts have been on studying endocrine-disrupting chemicals (EDCs), or commonly known as environmental hormones. These chemicals can imitate natural hormones and interfere not only the normal endocrinal, but also immune, reproductive and nerve systems in animals and humans. Some EDCs can be found in different water bodies or even in sewage-treatment works (STWs) effluents. The aims of this study were to investigate the effects of effluent from different sources on induction of vitellogenin (VTG) in the primary hepatocyte culture of Japanese medaka (Oryzias latipes) Because aquatic toxicology studies need to sacrifice a lot of animals, using cell culture can minimize the number of the animals used for different purposes. The medaka hepatocytes were cultivated in flasks with medium, and were exposed to 17-β estradiol and samples extracted from wastewater for 5days, 10days and 15days. The culture mediums were changed daily. VTG induction in hepatocytes was analyzed using Western blotting and quantified by the Bio-Rad’s image analysis system. The results showed that VTG induction by 17-β estradiol was dose-dependent after the 15-day exposure. Most of the influent samples from constructed wetlands could not induce vitellogenesis in cultured cells until exposed for 15 days. All of the effluent samples tested showed no VTG inducibility except those from An-Ping STW, and the responses were dependent with exposure time. Based on the results in this study, using Japanese medaka primary hepatic cell culture to investigate the effects of wastewater on induction of VTG was feasible, and at the same time, can be served as a alternative for in vivo study.
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