臺灣博碩士論文加值系統

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。
黃緯允(1998) 以單細胞電泳法分析哺乳類細胞游離輻射暴露後DNA的修補。國立陽明大學公共衛生研究所碩士論文。
Aravindan G.R., Bjordahl J., Jost L.K. and Evenson D.P. (1997). Susceptibility of human sperm to in situ DNA denaturation is strongly correlated with DNA strand breaks identified by single-cell electrophoresis. Experimental cell Reserch, 236: 231~237.
Balhorn R. (1982). A model for the structure of chromatin in mammalian sperm. J Cell Biol 93:298~305.
Belpaeme K., Cooreman K. and Kirsch-Volders M. (1998). Development and validation of the of the in vivo alkaline comet assay for detecting genomic damage in marine flatfish. Mutation Research, 415: 167~184.
Fairbairn D.W., Olive P.L. and O’Neill K.L. (1995). The comet assay: a comprehensive review. Mutation Research, 339:37~59.
Gajendiran N., Tanaka K. and Kamada N. (2000). Comet assay to sense neutron “fingerprint”. Mutation Research, 452: 179~187.
Garner D.L., Thomas C.A., Joerg H.W., Mel Dejarnette J. and Marshall C.E. (1997). Fluorometric assessments of mitochondrial function and viability in cryopreserved bovine spermatozoa. Biology of Reproduction, 57: 1401~1406.
Garner. D.L., Johnson L.A., Yue S.T. and Roth B.L. (1994). Dual DNA staining assessment of bovine sperm viability using SYBR-14 and Propidium Iodide. Journal of Andrology, 15: 620~629.
Gwo J.C., Kurokura H. and Hirano R. (1993). Cryopreservation of spermatozoa from rainbow trout, common carp and marine puffer. Nippon Suisan Gakkaishi, 59(5): 777-782.
Gwo J.C., Strawn K., Longnecker M.T. and Arnold C.R.（1991）. Cryopreservation of Atlantic croaker spermatozoa. Experimental of Zoology, 94:355-375.
Gwo J.C. (1995). Cryopreservation of oyster (Crassostrea gigas) embryo. Theriogenology, 43: 1163-1174.
Gwo J.C. (2000). Cryopreservation of aquatic invertebrate semen: a review. Aquaculture Research, 31:259-271.
Gwo J.C., Liou C.H. and Cheng C.H. (1996). Ultrastructure of the spermatozoa of the Pacific oyster Crassostrea gigas (Mollusca, Bivalvia, Ostreidae). Journal of Submicroscopy, Cytology and Pathology, 28: 239-244.
Hamamah S., Royere D., Nicolle J. C., Paquignon M. and Lansac J. (1990). Effects of freezing-thawing on the spermatozoon nucleus: a comparative chromatin cytophotometric study in the porcine and human species. Reprod Nutr Dev 30:59~64.
Hellman B., Vaghef H. and Borstrom B. (1995). The concepts and tail inertia in the single cell gal electrophoresis assay. Mutation Research, 336: 123~131.
Helma C. and Uhi M. (2000). A public domain image-analysis program for the single-cell gel-electophpresis (comet) assay. Mutation Research, 466: 9~15.
Holt W. V. (2000). Basic aspects of frozen storage of semen. Animal Reproduction Science, 62: 3~22.
Hughes C.M., Lewis S.E.M., McKelvey-Martin V.J. and Thompson W. (1997). Reproducibility of human sperm DNA measurements using the alkaline single cell gel electrophoresis assay. Mutation Research, 374:261~268
Ma S., Chang W. P. and Fang R. H.(1996). Measurement of radiation-induced DNA double-strand breaks in Human diploid fibroblasts from Keloid and normal skin by single-cell gel electrophoresis. Plastic and reconstructive surgery, 98: 821-826.
Mazur P. (1984). Freezing of living cell: mechanisms and implications. Am. J. Physiol, 247:C125-C142.
Mitchelmore C. L. and Chipman J. K. (1998). DNA strand breakage in aquatic organisms and the potential value of the comet assay in environmental monitoring. Mutation Research, 399: 135~147.
Moretti M., Villarini M., Scassellati-Sforzolini G., Santroni A. M., Fedeli D. and Falcioni G. (1998). Extent of DNA damage in density-separated trout erythrocytes assessed by the “comet” assay. Mutation Research, 397:353~360.
Ostling, O. and Johanson K.J. (1984). Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochemical & Biophysical Research Communications. 123(1): 291-298.
Pavlica M., Klobucar G.I.V., Mojas N., Erben R. and Papes D. (2001). Detection of DNA damage in haemocytes of zebra mussel using comet assay. Mutation Research, 490:209~214.
Ross G. M., McMillan T. J., Wilcox P. and Collins A. R. (1995). The single cell microgel electrophoresis assay (comet assay): technical aspects and applications Report on the 5th LH Gray Trust Workshop, Institute of Cancer Research, 1994 Mutation Research, 337: 57~60.
Salamon S. and Maxwell W. M. C. (1995). Review frozen storage of ram semen II.Causes of low fertility after cervical insemination and methods of improvement. Animal Reproduction Science, 38: 1~36.
Shen H.M. and Ong C.N. (2000). Detection of oxidative in human sperm its association with sperm function and male infertility. Free Radical Biology ＆ Medicine, 28:529~536
Singh, N.P., McCoy M. T., Tice R. R. and Schneider E.L. (1998a). A simple technique for quantitation of low levels of DNA damage in “comet” assay. Radiation Research. 122:86-94.
Watson P.F. (2000). The causes of reduced fertility with cryopreserved semen. Animal Reproduction Science, 60~61: 481~492.
Woods J. A.,O’Leary K. A., McCathy R. P. and O’Brien N. M. (1999). Preservation of comet assay slides: comparison with fresh slides. Mutation Research, 429:181~187.