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研究生:王怡惠
研究生(外文):Yi-Hui Wang
論文名稱:探討在氧化壓力下斑馬魚胚胎發育時期穀胱甘肽還原酶的表現與生化特性分析
論文名稱(外文):Expression of glutathione reductase is regulated by oxidative stress in zebrafish embryonic development and enzyme characteristic
指導教授:耿全福
指導教授(外文):Chuian-Fu Ken
學位類別:碩士
校院名稱:國立彰化師範大學
系所名稱:生物技術研究所
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:101
中文關鍵詞:活性氧分子谷甘胺酸還原酶
外文關鍵詞:ROSglutathione reductasecopper
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銅離子是生物體必需的微量元素,能維持細胞內多種酵素的功能,但若細胞內游離銅離子含量過高會引起氧化壓力造成傷害。本實驗探討魚類胚胎在高濃度銅離子環境中,胚胎發育所受影響及抗氧化酵素穀胱甘肽還原酶(GR)基因表現與所扮演的生理功能。以2.5-10 ppm的銅離子處理斑馬魚胚胎0-48 小時,活存率逐漸降低,48小時的半致死濃度為2.5ppm;以DCFH-DA分析銅離子處理過的胚胎,證實活性氧分子大量產生,且胚胎細胞蛋白質與脂質過氧化程度均明顯增加超過4倍,且胚胎內抗氧化分子穀胱甘肽(GSH)含量顯著降低至1%,進而證實GSH減少與胚胎內GR活性減少約50%有顯著關連。Whole-mount in situ hybridization證實此基因為全身性表現,最大量表現在胚胎頭部與眼睛,銅離子處理12hr後會誘導胚胎細胞增加GR基因的表現以抵抗氧化壓力。進一步探討斑馬魚重組GR酵素特性,由結果得知GR可於50℃加熱64分鐘後,仍保有55%的活性;而GR經過trypsin處理3小時後,還有87%活性,最適反應PH值介於7.4~11之間,顯示GR蛋白質耐鹼性但卻不耐酸,而經過12% SDS處理過後,GR就完全失去活性,另外經由酵素動力學分析結果可知GR的Km值為0.071mM,Vmax為0.042 s-1。


Copper (Cu) ion is an essential trace element for all organisms, but excess Cu ion exposure can produce oxidative stress and exert a variety of pathological effects. In this study, we treated zebrafish embryos with 2.5-10 ppm Cu ion for 48 h, the survival rate was decreased and the abnormal embryos were increased significantly. LC50 was about 2.5 ppm for Cu ion treated for 48hr. Reactive oxygen species (ROS) was elevated and the level of protein carbonylation and lipid peroxidation was also increased after Cu ion treatment. The GSH content and glutathione reductase (GR) activity were decreased significantly after Cu ion treatment. GR is a homodimeric flavoprotein that can reduce the GSSG to GSH and maintain high GSH/GSSG ratio in cells. The gene was mainly expressed on head and lens by whole-mount in situ hybridization. The gene expression was increased at Cu treated for 12 hour post fertilization (hpf). The results showed Cu can induce oxidative stress and regulated GR gene expression in zebrafish embryonic development. Recombinant GR can retained about 55% activity after heated at 50 ℃ for 64 minutes. It was resisted trypsin treatment for 3h, but was inactivated in SDS treatment. The best GR activity was shown between pH 7.4 to 11, The Michaelis constant (Km) values for NADPH is 0.071 mM, and the Vmax is 0.042 s-1.
目錄
章節 頁數
第一章、前言 9
(一) 活性氧分子(reactive oxygen species,ROS) 10
(二) Glutathione(GSH)介紹及其重要性 11
(三) Glutathione reductase (GR)之重要性 12
(四) 胚胎發育時期生物體內之抗氧化防禦機制 13
(五) 氧化壓力對生物體內抗氧化機制之影響 14
(六) 銅(Copper,Cu)介紹 14
(七) 研究目的 15
(八) 實驗流程 16
第二章、實驗材料 17
第三章、實驗方法 25
(1) 斑馬魚飼養與交配 25
(2) 斑馬魚的total RNA抽取 25
(3) 合成cDNA (MMLV kit) 25
(4) 聚合酶鏈反應(PCR) 26
(5) GR序列與載體黏合反應並轉形宿主細胞 27
(6) 勝任細胞之製備 28
(7) 質體之製備 29
(8) DNA 電泳 30
(9) 膠萃取 31
(10) DNA 定序 31
(11) 中量質體抽取 31
(12) 蛋白質結構模擬 32
(13) 蛋白質純化 33
(14) 透析 33
(15) 蛋白質定量 34
(16) GSH含量測定 34
(17) GR活性分析 35
(18) 銅離子對斑馬魚胚胎之處理 35
(19) Real-Time PCR 36
(20) 西方點漬法(Western blot) 37
(21) Whole-mount in situ hybridization 38
(22) 蛋白質誘導表現 42
(23) 生化特性分析 42
第四章、結果 45
(一) 斑馬魚GR序基因之選殖 45
(二) GR與其他物種比對 45
(三) GR蛋白質之結構分析 45
(四) GR蛋白質表現與純化 47
(五) GR酵素之特性分析 48
1. 熱穩定性分析 49
2. pH質耐受性分析 49
3. SDS耐受性分析 49
4. trypsin耐受性分析 49
5. 酵素動力學分析 50
(六) GR基因在斑馬魚胚胎發育時期表現 50
(七) GR在斑馬魚成體組織特異性分析 51
(八) 以銅作為氧化壓力下斑馬魚之存活率 51
(九) 銅處理對於斑馬於胚胎發育時期GR基因表現情形 52
(十) 斑馬魚胚胎發育時期GSH、GSSG與GSH/GSSG
ratio表現情形 53
(十一) 銅處理對於斑馬魚胚胎發育時期GSH、GSSG與GSH/GSSG
ratio的影響 54
(十二) 斑馬魚胚胎發育時期GR的表現 54
(十三) 銅處理對於斑馬魚胚胎發育時期GR活性的影響 54
第五章、討論 56
(一) 斑馬魚GR序基因之選殖與序列比對 56
(二)斑馬魚 GR蛋白質活性探討 56
(三) 重組斑馬魚 GR蛋白質特性分析 57
(四) GR基因在斑馬魚胚胎發育時期表現 58
(五) 銅處理對於斑馬於胚胎發育時期GR基因表現情形 59
(六) 斑馬魚胚胎發育時期GSH表現情形 60
(七) 斑馬魚胚胎發育時期GR活性的表現情形 61
(八) 未來展望 62
第六章、參考文獻 63
第七章、圖表 67
第八章、附錄 95

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