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研究生:吳鎮岩
研究生(外文):Tseng-Yen WU
論文名稱:彗星生物檢定法在水產生物上的應用-以牡蠣精子為例
論文名稱(外文):The application of Comet assay in aquatic invertebrate-oyster(Crassostrea gigas)sperm as an example
指導教授:郭金泉郭金泉引用關係
指導教授(外文):Jin-Chywan GWO
學位類別:碩士
校院名稱:國立海洋大學
系所名稱:水產養殖學系
學門:農業科學學門
學類:漁業學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
中文關鍵詞:彗星生物檢定法凍結保存無脊椎動物核酸單細胞洋菜膠電泳法精液牡蠣環境監測
外文關鍵詞:Comet assaycryopreservationInvertebratenucleic acidSingle cell gel electrophoresissemenoysterEnvironmental monitor
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慧星生物檢定法(comet assay)乃1984年由Ostling與Johnson氏發展出來,特性是以光學顯微鏡目視的方式,能簡易、經濟、方便、快速的檢測出每一個單獨細胞DNA的受損情形。之後經過多次的改良,此方法已廣泛運用於檢定哺乳類動物包括人類細胞受到外來物質(如放射線、化學藥品等)影響時DNA所受的傷害。我們改良應用於哺乳類動物之慧星生物檢定法,試圖使此檢定法也能適用於檢測水產生物細胞DNA所受的損害。我們以水產生物牡蠣精子為例,比較不同滲透壓、pH、電壓、電流強度、染色劑種類等因子對牡蠣精子之影響,找出適當的檢定方法。探討凍結保存是否會對於細胞核的DNA造成傷害,以新鮮牡蠣與以10﹪DMSO為抗凍劑凍結保存後的精子以及最好凍結條件(10﹪DMSO為抗凍劑)與最壞凍結條件(10﹪PEG為抗凍劑)作比較,驗證慧星生物檢定在水產生物牡蠣精子的實際應用成效。

The comet assay is a simple, sensitive, inexpensive and rapid technique for detection of DNA damage in individual cells. After being inverted by Ostling and Johansson in 1984. The assay was improved several times. Now this assay is usually applied in testing DNA damages by external genotoxicity (ex. Radiation and chemical) in mammal include human cell. I modified the comet assay, used in mammalian, and tried to inspect DNA damages in invertebrate. I use oyster sperm as an example, and try to find the optimize way to examine the influence on oyster sperm by compare different osmosis、pH、voltage、current intensity and stain. Confer to if the cryopreservation induces DNA damage. Use the fresh oyster in 10﹪DMSO to serve as the best frozen condition and 15﹪PEG to serve as the worst frozen condition for verifying the actuality in oyster sperm. The result in detection of DNA damage of oyster sperm, that we find the comet parameter divides into two different groups. However the best and worst condition is unable to discriminate.

壹.前言…………………………………………………………………………….1
I.凍結保存的原理………………………………………………..1
II.凍結保存的意義與價值…………………………………………………2
III.DNA傷害………………………………………………………………...2
III-1.彗星生物檢定法………………………………………………………3
III-2.單細胞電泳分析的原理………………………………………………3
貳.材料與方法……………………………………………………..4
I.種貝的來源與處理…………………………………………….……….4
II.配子的收集…………………………………………………………………………4
III.牡蠣精子的凍結保存………………………………………………………...……4
IV.彗星生物檢定法……………………………………………………………...……5
VI.牡蠣精子彗星生物檢定法最佳化………………………………...……5
VI-1.低凝固點洋菜膠(LMP)的備製……………………..….…………5
VI-2.溶解緩衝液(Lysis buffer)的選擇………………………………5
VI-3.電泳的時間與電量………………………………………..………..6
VI-4.染劑的選擇………………………………………………..………..6
VI-5.玻片保存…………………………………………………..………..6
VII.影像選取的標準…………………………………………….…..…..7
VIII.影像處理與分析………………………………………….………...7
IX.參數之選擇…………………….....……………………….………..7
X.統計分析…………………………………………………….…….……..8
參.結果…………………………………………………………………….…9
I.步驟之最佳化…………………………………………………………….9
I-1滲透壓實驗……………………………………………………………..9
I-2溶解緩衝液實驗…………………………………………………….….9
I-3.電泳實驗………………………………………………………………..9
I-4.染劑實驗………………………………………………………………..9
I-5.玻片保存實驗………………………………………………………….10
II.資料分析………………………………………………………………..10
肆.討論……………………………………………………………………..12
伍.謝辭……………………………………………………………………..15
陸.參考文獻…………………………………………………………………16
柒.圖………………………………………………………………………..20
捌.表………………………………………………………………………..29

陳銓汶 (2001) 水產無脊椎動物牡蠣與九孔精液之凍結保存。國立海洋大學水產養殖學系碩士論文。
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