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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/34650

Title: 鎘藉由ROS訊息途徑抑制斑馬魚胚胎中DNA錯誤配對辨識蛋白MSH2及MSH6之基因表現
Cd down-regulates the gene expression of DNA mismatch recognition proteins MSH2 and MSH6 in zebrafish (Danio rerio) embryos via ROS-dependent signaling pathway.
Authors: Shih-Tsung Sung
宋世聰
Contributors: NTOU:Institute of Bioscience and Biotechnology
國立臺灣海洋大學:生物科技研究所
Keywords: ;錯誤配對修補;斑馬魚
Cadmium;mismatch repair;zebrafish
Date: 2013
Issue Date: 2013-10-07T02:44:50Z
Abstract: 真核生物之 DNA 錯誤配對修復系統 (mismatch repair;MMR) 是由MutS homolog 2 (MSH2)/MSH6 構成的 MutSα複合體辨識單一鹼基配對錯誤,與小型寡核苷酸環。鎘為具致癌性的重金屬,且已知鎘可能透過引起活性氧化物 (Reactive oxygen species,ROS) 之過度增加,與抑制 DNA 修補作用而產生致癌性。先前本實驗室已發現非致死濃度的鎘可抑制斑馬魚胚胎中 msh2/6 之基因表現。因此本論文接著探討鎘是否透過 ROS 與磷酸化的途徑抑制 msh2 及 msh6 表現。將受精1小時後 (hpf) 之斑馬魚胚胎處理非致死濃度之1~5μM氯化鎘,依據 malondialdehyde (MDA) 含量之明顯上升得知鎘於此濃度範圍均引起胚胎內氧化壓力上升。由全覆式原位雜合法也顯示以抗氧化劑10µM N-acetylcysteine (NAC)或3µM butylhydroxytoluene (BHT)於加鎘前處理半小時可回復鎘對 msh2/msh6 表現之抑制效應。另外直接處理 100-200μM 之超氧陰離子產生劑 paraquat 和過氧化氫同樣會抑制胚胎組織中 msh6 的表現。即時定量 PCR (QPCR)分析結果也得知抗氧化劑 NAC 與 BHT 可回復鎘抑制之 msh2/6 和Cu/Zn-SOD 基因表現。原位雜合與 QPCR 也發現於加鎘前以 1~3nM 的廣效性蛋白激脢抑制劑 staurosporine 處理半小時,並未見到 msh2/msh6 表現有明顯回復。實驗結果得知鎘會透過產生 ROS 使 msh2/6 之表現下降,但ROS 可能並非藉由影響下游磷酸化影響 msh2/msh6 之基因活性。
In eukaryotes, DNA mismatch repair (MMR) of simple base mismatches and small insertion-deletion loops is initiate by the binding of the MutS homolog 2 (MSH2)-MSH6 heterodimer to mismatched DNA. Cadmium (Cd) is a carcinogenetic heavy metal that has been recognized as a human carcinogen. Generation of reactive oxygen species (ROS) and inhibition of DNA repair have been proposed as major factors of Cd-induced genotoxicity. This study explored if Cd-induced oxidative stress down-regulated msh2/msh6 gene expression and whether kinase activities were involved in the process. Sublethal levels of Cd was found to induce oxidative stress in 10hpf zebrafish embryo after a 9-h exposure based on the overproduction of MDA. Whole mount in situ hybridization revealed the down-regulated msh2/msh6 expression in embryos exposed to paraquat, a reactive oxygen species (ROS)-generating herbicide, at 100 to 200 μM or 200 μM hydroxyl peroxide. Pretreatment of embryos with antioxidants butylhydroxytoluene (BHT), or N-acetylcysteine (NAC) at 3–10 μM for 0.5h restored Cd-suppressed msh2/msh6 expression. Furthermore, protective effects of antioxidants on Cd-suppressed msh2/msh6 and Cu/Zn superoxide dismutase (Cu/Zn-SOD) mRNA production were detected by real-time RT-PCR after a 9-h Cd exposure. Hence, Cd at sublethal levels down-regulates msh2/msh6 expression primarily via ROS as signaling molecules.The expression of msh2/msh6 mRNA in zebrafish embryos was unaffected by the broad-spectrum kinase inhibitor staurosporine at 1 to 3 nM after a 9-h exposure. Pretreatment of embryos with staurosporine of 1-3 nM for 0.5h failed to protect against Cd-induced MSH gene inhibition, suggesting that Cd-induced oxidative stress did not down-regulate MSH gene activities through inhibition of downstream kinases.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0019936014
http://ntour.ntou.edu.tw/handle/987654321/34650
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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