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研究生:黃冠銘
研究生(外文):Kuan-Ming Huang
論文名稱:重金屬汞與鎘對斑馬魚胚胎中DNA錯誤配對 (MMR) 辨認蛋白MutS homolog 2及6 (MutSα) 基因表現之影響與可能機制
論文名稱(外文):Effects of cadmium and mercury on the gene expressions of DNA mismatch (MMR) recognition protein, MutS homolog 2&6 (MutSα) in zebrafish (Danio rerio) embryo and possible mechanisms
指導教授:許濤許濤引用關係
指導教授(外文):Todd Hsu
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:106
中文關鍵詞:DNA錯誤配對修復系統活性氧
外文關鍵詞:cadmiummercurymismatch repairROS
相關次數:
  • 被引用被引用:4
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  • 下載下載:40
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DNA錯誤配對修復系統 ( mismatch repair ; MMR) 可移除單一鹼基配對錯誤及因未配對的鹼基形成寡核苷酸環(insertion/deletion loops ; IDLs)。真核生物之辨認蛋白MutSα (MSH2/MSH6),主要辨認單一的核酸錯誤配對如G-T配對。鎘為致癌重金屬且本實驗室先前已發現鎘抑制斑馬魚胚胎MMR辨認蛋白基因表現。因此本論文接續前人之研究探討鎘抑制MMR基因表現之可能機制及同一族之二價汞對MMR辨認蛋白基因表現之影響。由QRT-PCR及錯誤配對結合分析,鎘於亞致死濃度處理斑馬魚胚胎9小時對msh2/6及蛋白表現有明顯性抑制,而汞只有中等程度抑制。以全覆式原位雜交法也證實重金屬處理對胚胎組織中msh6表現造成抑制。鎘也抑制銅鋅超氧歧化酶(Cu/Zn-SOD)基因表現,但對過氧化氫酶(Catalase)基因表現似乎沒有影響。此外,10 μM的D-甘露醇、N-乙醯半胱胺酸及butylhydroxytoluene (BHT)可阻斷3 μM鎘對msh2/6表現之抑制,表示活性氧(ROS)如超氧陰離子自由基參與鎘誘導之基因下調效應。過氧化氫對msh2/6及SOD表現無顯著影響。人類msh6轉錄需藉由Sp1協助,但胚胎經鎘與汞處理9小時對Sp1的DNA結合活性及Sp1蛋白無顯著抑制。因此,本研究認為鎘抑制msh2/6表現是透過ROS產生;也發現汞會干擾MMR的辨識步驟。
Cadmium (Cd) is a carcinogenic heavy metal which may exert its carcinogenicity by decreasing the efficiency of DNA repair. Our previous studies indicated the ability of Cd ion to down-regulate the gene expressions of MutS homolog 2 (MSH2) and MSH6 that initiates DNA mismatch repair (MMR) of simple mispairs and small insertion-deletion loops in vertebrates using zebrafish (Danio rerio) embryo as a model organism. This study explored the mechanisms of Cd-induced inhibition of msh2/msh6 activities and the effects of mercuric ion on the two genes. Cd at sublethal and low toxic concentrations significantly inhibited msh2/msh6 expressions in zebrafish embryos after a 9-hr exposure as shown by QRT-PCR and mismatch binding assay, while Hg imposed only medium inhibitory effects. Inhibition of msh6 mRNA production in the tissues metal-treated embryos was confirmed by whole mount in situ hybridization. Cd exposure also suppressed the gene expression of Cu-Zn superoxide dismutase (SOD), but the expression of catalase gene was hardly affected by this metal. Moreover, the inhibitory effects of 3 μM Cd on msh2/msh6 expressions were totally reversed in the presence of 10μM D-mannitol, N-acetylcysteine, or butylhydroxytoluene, reflecting the participation of reactive oxygen species (ROS), superoxide anion radical possibly the major one, in Cd-induced gene down-regulation. Gene expressions of msh2/msh6 and SOD were generally unaffected by hydrogen peroxide. The transcription of human msh6 is dependent on the Sp1 activity, but Cd or Hg exposure barely disturbed the DNA binding activity of Sp1 factor or Sp1 protein synthesis in embryos after a 9-hr exposure. Hence, Cd is believed to inhibit msh2/msh6 expressions via the production of ROS and this study also showed the potential of Hg to disturb the recognition step of MMR.
謝 誌 I
摘 要 II
Abstract III
第一章 前言 1
1.1 DNA 錯誤配對修復系統( mismatch repair ; MMR) 1
1.2重金屬-汞(Mercury ; Hg) 5
1.3重金屬-鎘(Cadmium ; Cd) 8
1.4斑馬魚(Zebrafish, Daino rerio)胚胎發育研究之動物模式 12
1.5研究動機及目的 13
名詞縮寫 16
第二章 實驗材料 ( Materials ) 18
2.1藥品配製 21
2.2實驗儀器設備 28
第三章 實驗方法 ( method ) 29
3.1 斑馬魚之飼養 29
3.2 RNA及DNA片段完整性 32
3.3 反轉錄酶-聚合酶鏈鎖反應 (Reverse transcriptase-polymerase chain reaction ; RT-PCR) 33
3.4 即時定量聚合酵素連鎖反應 (Quantitative Real-time PCR; QRT-PCR) 34
3.5 RNA全覆式原位雜交 (RNA whole-mount in situ hybridization) 35
3.6 蛋白質定量- BCA Protein Assay 42
3.7 凝膠電泳阻滯試驗 ( Electrophoretic mobility shift assay; EMSA ) 43
3.8 SDS-PAGE蛋白質分析 45
3.9 西方墨點法 ( Western Blot ) 45
第四章 結果 47
4.1 受精24小時內斑馬魚早期胚胎中msh2與msh6之表現 47
4.2 鎘與汞對斑馬魚胚胎DNA錯誤配對辨識活性之影響 48
4.3 鎘與汞對斑馬魚胚胎轉錄因子Sp1辨識活性之影響 48
4.4 鎘對斑馬魚胚胎中Sp1蛋白質表現之影響 49
4.5 汞對斑馬魚胚胎中MSH6及Sp1蛋白質表現之影響 50
4.6 重金屬-汞及鎘和過氧化氫對斑馬魚早期胚胎發育存活率之影響及LC50 50
4.7 鎘處理4及9小時對斑馬魚早期胚胎中msh2、msh6、Cu/Zn-SOD及Catalase之影響 51
4.8 汞處理4及9小時對斑馬魚早期胚胎中msh2、msh6、Cu-Zn-SOD及Catalase之影響 52
4.9 氧化劑(過氧化氫)處理11小時對斑馬魚早期胚胎中msh2、msh6及Cu-Zn-SOD之影響 53
4.10 抗氧化劑與鎘處理9小時對斑馬魚早期胚胎中msh2、msh6、Cu-Zn-SOD及Catalase之影響 53
4.11 全覆式原位雜交法分析早期斑馬魚胚胎中經重金屬之不同濃度處理後msh6 mRNA之影響 54
第五章 討論 55
第六章 參考文獻 58
結果圖 70
附圖 A 斑馬魚不同時期之胚胎發育圖 (Kimmel et al., 1995) 94
附圖 B 錯誤配對修復系統 (Mismatch repair ; MMR) (Stojic et al., 2004) 96
Table 1. Sequences of primers for semiquantification RT-PCR 97
Table 2. Sequences of primers for quantitative real-time RT-PCR 98
Table 3. Sequences of biotin-labelled oligonucleotide probes for EMSA 99

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