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研究生:蔡惠婷
研究生(外文):Huei-Ting Tsai
論文名稱:砷與鎘對發育中斑馬魚DNA錯誤配對辨識蛋白MutS homolog 2及6(MSH2&6) 基因表現之影響
論文名稱(外文):Effects of Arsenic and Cadmium on the gene expressions of DNA mismatch recognition proteins, MutS homolog2 &6 (MSH2&6) in developing zebrafish (Danio rerio)
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:2010
畢業學年度:98
語文別:中文
論文頁數:62
中文關鍵詞:MSH2MSH6錯誤配對修補機制
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DNA 配對錯誤修復系統 (DNA mismatch repair, MMR),存在於原核及真核生物中,為修補DNA複製或交換過程中產生之鹼基錯誤配對之重要機制。
於真核細胞內,單一配對錯誤或小環嵌入與刪除寡核苷酸(insertion-deletion loops, IDLs) 由MSH2與MSH6形成的複合體MutSα行辨識結合;而MSH2與MSH3形成的複合體MutSβ則負責大環IDLs。鎘及砷皆為可引起DNA斷裂之致癌性重金屬,本論文主要探討氯化鎘及亞砷酸鈉處理斑馬魚胚胎後對MSH2及MSH6基因表現之影響,以了解鎘與砷之遺傳毒性機制。RT-PCR結果顯示MSH2及MSH6 mRNA於受精後1 ~ 25小時 ( hour post fertilizatioin, hpf ) 之斑馬魚胚胎中呈同步表現,mRNA含量於1 hpf胚胎中甚低,但在2 hpf胚胎中則急遽上升。此兩種基因持續表現在後期胚胎中,僅在12 hpf斑馬魚中表現量明顯降低。而當1 hpf胚胎經0.5至3.0 μM氯化鎘處理後,MSH6較 MSH2對鎘更為敏感。統計分析結果顯示,經鎘濃度0.5至3.0 μM處理之MSH6 mRNA呈有意義的下降,相對地MSH2 mRNA則需至3μM才可見有意義的下降;由即時定量PCR測定後發現,胚胎經鎘處理後此兩基因表現皆受抑制,其中3.0 μM Cd處理11小時候其表現量減少50%以上。另外,MSH2及MSH6 mRNA於1 hpf之RT-PCR中表現量雖低,但全覆式原位雜合法在一個細胞期即為可見。而透過全覆式原位雜合法,經鎘處理之12 hpf 胚胎亦發現與RT-PCR實驗類似的情形,在鎘濃度0.5至3.0 μM處理下,MSH6 mRNA的表現被抑制,而MSH2 mRNA則是在鎘濃度3 μM時才被抑制。但即時定量PCR發現砷並無明顯基因抑制作用,故本論文僅進一步以EMSA及西方點墨法探討鎘抑制 MSH基因表現之蛋白質層級影響。除此之外,經EMSA及Western分析其MSH6活性及蛋白質表現量,隨鎘濃度的增加而減少其表現量。實驗結果顯示,鎘可能藉著抑制MMR系統中MSH6辨識蛋白基因表現,而導致遺傳物質不穩定造成基因毒性。由於機制尚未清楚,仍有待進一步證實與深入探討。

DNA mismatch repair (MMR) identifies replication errors and damaged bases in DNA and takes corrective actions to preserve genomic integrity. MMR plays an important role in maintaining genome stability by primarily correcting both base-base mismatches and insertion/deletion (ID) mispairs generated during DNA replication. MutS homolog 6 (MSH6) is the major mismatch contacting component of the MSH2-MSH6 heterodimeric complex (MutSα) that mediates DNA mismatch repair (MMR) of simple mispairs and small insertion-deletion loops in eukaryotes. Arsenic (As) and cadmium (Cd) are widespread in the environment and both heavy metals are classified by the IARC as human carcinogens. This study examined the potential of Cd and As to disturb the gene expression of MSH6 in vertebrates using zebrafish (Danio rerio) embryo as a model organism. Semiquantitative RT-PCR indicated that both msh2 and msh6 expressions were suppressed in zebrafish embryos at 1 h post fertilization (hpf), then drastically up-regulated in 2 hpf embryos and remained active in 3 to 25 hpf embryos. In the presence of a constitutive β-actin expression, exposure of 1 hpf embryos to sublethal concentrations of CdCl2 at 0.5 to 3.0 μM for 5 or 11 h caused a time and concentration-dependent down-regulation of msh2 and msh6 transcription as shown by semiquantitative RT-PCR. Cd failed to inhibit msh2 transcription except at 3μM, reflecting the higher sensitivity of msh6 than msh2 transcription to Cd. Quantitiative real-time PCR also showed the higher sensitivity of msh6 than msh2 transcription to Cd and Cd at 0.5 μM significantly inhibited both msh6 and msh2 transcription after a 11-h exposure. Whole mount in situ hybridization showed a wide distribution of 12 hpf embryos and Cd treatment induced a general suppression of msh6 expression in zebrafish tissues. Because As at 25 to 400 μM was unable to impose a significant inhibitory effect on both msh2 and msh6 expressions after analysis by real-time PCR, this research only explored the effect of Cd on MSH gene expressions at the protein level. Cd-induced down-regulation of msh6 transcription in zebrafish embryos paralleled with reduced levels of MSH6 protein synthesis and MSH6-mediated G-T mismatch binding activities identified by band shift assay using an anti-human MSH6 antibody. The results of this study revealed the inhibition of Cd on MSH6 expression at both mRNA and protein levels and this mechanism may play a role in Cd genotoxicity. Trivalent As was found to be less active than Cd in affecting MSH6 expression. The mechanisms of Cd-induced suppression of msh2 and msh6 expression await further investigation.
摘要 ii
Abstract iv
目錄 vi
圖目錄 ix
第一章 前言 1
1-1重金屬鎘 1
1-2重金屬砷 2
1-3 DNA錯誤配對修復系統 ( DNA mismatch repair;MMR ) 3
1-4重金屬鎘與砷對DNA修補作用之影響 4
1-5 MMR與疾病 5
1-6 斑馬魚之胚胎發育研究模式 5
1-7 論文研究目的 6
1-8 實驗架構 7
第二章 研究材料與設備 8
2-1研究材料 8
2-1-1 實驗動物 8
2-1-2 菌種及質體DNA 8
2-1-4 半定量PCR 引子序列 (購自明欣生物科技有限公司) 9
2-1-5 即時定量PCR 引子序列 (購自明欣生物科技有限公司) 10
2-2 實驗藥品配製 11
2-2-1 斑馬魚粗萃取液之藥品配製 11
2-2-3 凝膠阻滯電泳相關之藥品配方 12
2-2-4 西方點墨法 Western Blot相關配方 13
2-2-6 DNA 電泳之相關藥品 15
2-2-7 微量質體DNA 萃取之藥品配製 15
2-2-8 質體轉型之藥品配方 16
2-2-9 RNA 凝膠電泳之藥品配方 16
2-2-10 RNA 全覆式原位雜交之藥品配方 16
2-3 儀器設備 18
2-4 實驗方法 ( method ) 19
2-4-1 斑馬魚飼養與受精胚胎之收集 (Westerfield, 1993) 19
2-4-2斑馬魚胚胎萃取液之製備 20
2-4-3 蛋白質濃度測定- BCA Protein Assay 試劑 Kit 20
2-4-4 凝膠電泳阻滯試驗 ( Electrophoretic mobility shift assay, EMSA ) 21
2-4-5 西方轉漬法實驗 ( Western Blot ) 23
2-4-6 斑馬魚胚胎RNA萃取 23
2-4-7 RNA定量 24
2-4-8 反轉錄酶-聚合酶鏈鎖反應 (Reverse transcriptase-polymerase chain reaction, RT-PCR) 25
2-4-9 半定量 PCR (semi-quantification PCR) 操作 25
2-4-10 即時定量聚合酵素連鎖反應 (Quantitative Real-time PCR) 26
2-4-11 核酸電泳分析 27
2-4-12 RNA全覆式原位雜交 (RNA whole-mount in situ hybridization) 探針(probe)製作前製備 28
2-4-13全覆式原位雜交RNA探針(RNA probe)之合成 31
2-4-14 RNA 全覆式原位雜交 (RNA whole-mount in situ hybridization) 32
第 三 章 結 果 36
3.1重金屬鎘及砷對斑馬魚早期胚胎發育存活率之影響 36
3.2 斑馬魚早期胚胎發育中MSH2與MSH6之基因表現 36
3.3半定量 PCR 分析鎘對斑馬魚胚胎MSH2及MSH6基因表現之影響 37
3.4 相對定量PCR分析鎘及砷對斑馬魚胚胎MSH2及MSH6基因之影響 38
3.5 全覆式原位雜合分析斑馬魚早期胚胎中MSH2 mRNA組織表現分佈 38
3.6 鎘對於斑馬魚胚胎組織中msh2及msh6表現之影響 38
3.7 鎘對斑馬魚胚胎DNA錯誤配對辨識活性之影響 39
3.8 鎘對斑馬魚胚胎中MSH6蛋白質表現之影響 40
第四章 討 論 41
第五章 Refernse 44
附圖1、 斑馬魚胎胚胎不同時期生長型式。 61
附圖2、錯誤配對核酸修補 (mismatch repair; MMR) 模式。 62

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