臺灣博碩士論文加值系統

中文摘要
細胞凋亡是生物體發育（development）及更生（remodel）上必經的過程，而自由基則可能參與了細胞凋亡的過程。本研究主在觀察山羊乳腺在乾乳時期細胞凋亡與自由基清除的關係。試驗將山羊乳腺的生理時期分為四期，分別為末期（late）（乾乳前三週至乾乳）；乾乳後1週（involution 1）；乾乳後2週（involution 2）；及高峰（peak）（分娩後4至6週）。再將乳腺分泌物分成去掉細胞的乳漿和細胞兩部分，分別觀測比較此兩部分在四個時期中乳漿內及細胞內的抗氧化酵素：超氧化物歧化 （SOD）、麩胱甘過氧化物（GPx）的活性以及脂質氧化值（TBARS），同時再觀察乳腺分泌物中細胞的型態及DNA的斷裂情形。
試驗結果顯示--以光學顯微鏡觀察乳腺分泌物中的細胞形態，發現多形核嗜中性球（polymorphonuclear leukocyte；PMN）佔細胞數目的大多數，PMN的直徑大小約在10-15μm之間。經由免疫組織化學染色法來確認乳腺上皮細胞，發現上皮細胞的直徑大小約在25-50μm之間，明顯大於其他乳腺分泌物中的細胞，另外乾乳時可以發現乳腺分泌物中的細胞具有空泡，同時在乳腺分泌物中細胞的DNA電泳圖上可發現階梯狀的DNA斷片，而在泌乳高峰則無此現象。鏡檢乳腺分泌物中的細胞數目，發現乾乳後1週與2週時，細胞數目分別是56.8×105/ml與52.6×105/ml顯著高於末期（28.2×105/ml）與高峰（3.8×105/ml）（P＜0.05），但是乾乳後1週與2週兩期之間的細胞數目則無明顯差異（P＞0.05）。乳腺分泌物中細胞DNA的含量及DNA斷片的百分比，在乾乳2週時分別為8.89 mg/100ml及76﹪，顯著高於其他三個時期（P＜0.05）。另外在抗氧化酵素活性及過氧化程度的測定方面--乳漿內：SOD的活性在乾乳第1週為1.07 U/ml顯著高於其他三期（P＜0.05），GPx的活性在末期高達0.31 U/ml，此時活性為最高（P＜0.05），TBARS的測定值則在乾乳的第2週顯著高於另外三期（P＜0.05），其值為3.34μM；在細胞內方面：在乾乳後1週與2週時SOD的活性各別為148.27 U/mg DNA與135.65 U/mg DNA，相對於其他二期為低（P＜0.05），GPx在乾乳後1週與2週的活性分別為99.21 U/mg DNA及90.29 U/mg DNA，顯著低於另外兩期（P＜0.05），但是乾乳後1週與2週兩者之間GPx活性的差異並不顯著（P＞0.05），TBARS值在四個分期的變化則無顯著差異（P＞0.05）。
在山羊乳腺分泌物中細胞內各項測定值之間的相關方面：TBARS與DNA的斷片百分比呈顯著正相關r＝0.33（P<0.05）；TBARS值與DNA含量之相關係數為r=0.40（P<0.01），呈極顯著之正相關；SOD活性與GPX活性之正相關性很高（r=0.99， P<0.01）；抗氧化酵素SOD及GPX的活性與DNA含量有極顯著的負相關（r= -0.45及r= -0.44，P<0.01）；抗氧化酵素SOD與GPX的活性越高，DNA的斷片百分比越低（r= -0.38及r= -0.39，P<0.05）。乳漿的各項測定值相對於羊乳中細胞內各項測定值的相關：乳漿中TBARS值對於細胞內TBARS值與DNA斷裂百分比有顯著正相關（r= 0.30及r= 0.32，P<0.05），以及乳漿中SOD的活性對於DNA斷裂百分比有顯著正相關（r= 0.47，P<0.05）。
根據以上的結果顯示：乾乳時期乳腺分泌物中細胞的DMSCC、DNA總量及DNA斷裂百分比有顯著的上升，同時期乳腺分泌物中的細胞亦發生細胞凋亡的現象，乾乳期乳漿中過氧化程度（TBARS）明顯高於其他時期，同時細胞內SOD及GPx的活性均較末期與高峰為低，推測極可能是自由基對細胞膜產生了氧化性破壞後，誘發產生細胞凋亡的過程。

英文摘要
Apoptosis is a form of cell death which provides the organism with both the capability to remove specific cells during development and a way to continuously turnover cells in tissues. Oxidants such as oxygen free radicals may be essential biochemical intermediates in the progression of apoptosis. In the present study, involution of goat mammary gland following dry off was used as a model for researching apoptosis and antioxidants events. Mammary secretions were collected from goats in four different physiological stages：3 to 1 weeks before dry off（late）, 1 week following dry off（involution 1）, 2 weeks following dry off（involution 2）and 4 to 6 weeks after parturition（peak）. Cellular and non-cellular liquid parts of mammary secretion were separated（500×g, 10 min）and were used for assaying antioxidative enzymes superoxide dismutase（SOD）and glutathione peroxidase（GPx）and measurement of lipid peroxidation as represented by concentration of thiobarbituric acid reactive substances（TBARS）. Cell type and morphology as well as the manner and extent of DNA degradation were also observed.
Polymorphonuclear leukocyte（PMN）with an average diameter 10-15 μm was the major cell type in all stages. Mammary epithelial cell was identified immunohistochemically by fluorescence microscopy. Vacuoles and oligonucleosomal DNA laddering in ethidium bromide-stained gel indicated that cells loss by apoptosis occurred starting in late stage and most prominently during involution, but apparent not during peak stage. Direct microscopic somatic cell counts（DMSCC）for 1（56.8×105/ml）and 2（52.6×105/ml）weeks following dry off were significantly higher than those for late（28.2×105/ml）and peak（3.8×105/ml）stages. The contents of total cellular DNA（8.89mg/dl）and percentages of cellular DNA fragmentation（76﹪）during involution were higher than those of the rest stages. For non-cellular liquid of mammary secretion, SOD activity was the highest in stage involution 1（1.07 U/ml）, GPx was the highest during late stage（0.31 U/ml）. Concentrations of TBARS was the highest in stage involution 2. For the cellular part of mammary secretion, activities of SOD and GPx were significantly lower during involution than late and peak stages. No differences in concentrations of TBARS was observed for these four stages.
Concentrations of cellular TBARS was highly correlated with the percentages of DNA fragmentation（r＝0.33）and cellular DNA content（r＝0.40）. Cellular activities of SOD and GPx were negatively correlated with percentages of DNA fragmentation（r＝-0.38 ＆ -0.39）. Concentrations of TBARS and SOD activity in non-cellular mammary secretion was highly correlated with percentages of DNA fragmentation（r＝0.32 ＆ 0.47）.
In summary, during 1 to 2 weeks following dry off, there were significant elevation of DMSCC, total cellular DNA content, percentages of DNA fragmentation and concentrations of TBARS in mammary secretion. Meanwhile, activities of cellular SOD and GPx in mammary secretion were significantly depressed. Appearance of DNA laddering demonstrates the progression of apoptosis during involution. Finally, concentrations of TBARS in mammary secretion is a good index of mammary involution, and the lowering of antioxidative enzyme activities in cell of mammary secretion during involution probably caused DNA fragmentation therefore suggests a role of oxygen free radical in apoptosis of mammary involution.

目錄
壹、中文摘要 1
貳、前言 3
參、文獻檢討 4
一、泌乳週期 4
二、乳汁中的體細胞 4
三、細胞週期 4
四、細胞死亡的形式 4
五、凋亡的形態變化 7
六、凋亡的生化變化 7
七、自由基及活性氧 10
八、自由基對生理功能之影響 10
a 自由基及活性氧對生理功能之貢獻 10
b 自由基造成的傷害 12
c 脂質過氧化（Lipid peroxidation） 12
d 蛋白質的氧化傷害 15
e 對核酸造成的氧化傷害 16
九、生物體內的抗氧化系統 17
a 維生素 17
b 金屬螯合劑 18
c 代謝物 18
d 抗氧化酵素 18
肆、材料與方法 21
一、試驗動物 21
二、飼養管理 21
三、乳腺分泌物的採集 21
四、乳漿與細胞的分離方法 22
五、顯微鏡直接計數體細胞 22
六、上皮細胞的免疫組織染色觀察 23
七、乳漿中抗氧化酵素及過氧化程度的測定 23
a TBARS（thiobarbituric acid reactive substance）的測定 23
b 抗氧化酵素SOD活性的測定 23
c 抗氧化酵素GPx活性的測定 23
八、細胞溶解液的製備 25
九、細胞內抗氧化酵素及過氧化程度的測定 27
a TBARS（thiobarbituric acid reactive substance）的測定 27
b 抗氧化酵素SOD活性的測定 27
c 抗氧化酵素GPx活性的測定 27
十、細胞內DNA 斷片（fragmentation）的百分比 27
十一、DNA的定量 30
十二、細胞內DNA 的萃取 30
十三、DNA電泳 30
十四、統計分析 32
伍、結果與討論 33
一、乳腺分泌物中細胞形態的觀察 33
二、乳腺分泌物中細胞的數目 33
三、乳腺分泌物中細胞內DNA總量的分析 39
四、乳腺分泌物中細胞內DNA的完整性 39
五、不含細胞之乳漿內抗氧化酵素活性與脂質過氧化程度 42
a SOD的活性 42
b GPx的活性 45
c TBARS值 45
六、乳腺分泌物中細胞內抗氧化酵素活性與脂質過氧化的程度 48
a SOD ＆GPx的活性 48
b TBARS值 52
七、山羊乳腺分泌物中各項測定值之相關 54
a 細胞內各項測定值之間的相關 54
b 乳漿的各項測定值相對於細胞內各項測定值之間的相關 54
八、乳腺退化過程與自由基及過氧化所致細胞凋亡的可能相關 57
陸、結論 59
柒、參考文獻 60
捌、英文摘要 68
圖次
Fig1、Mammary growth during the lactation cycle: a generalized scheme 5
Fig2、Diagram of the cell cycle 8
Fig3、Sequence of ultrastructureal changes in apoptosis and necrosis 9
Fig4、Schematic diagram of internucleosomeal DNA fragmentation in apoptosis……..11
Fig5、The interacting mechanisms of cell injury by oxidative stress……………………13
Fig6、Relationship between nutrients and cellular antioxidant systems 20
Fig7、Standard curve of thhiobarbituric acid reactive substance (TBARS) concentration in milk 24
Fig8、Standard curve of superoxide dismutas (SOD) activity in milk determined from inhibition of 8 mM pyrogallol autooxidation 26
Fig9、Standard curve of thiobarbituric acid reactive substance (TBARS) concentration in cell of mammary secretion 28
Fig10、Standard curve for cellular superoxide dismutase (SOD) activity determined from inhibition of 8 mM pyrogallol autooxidation 29
Fig11、Standard curve of DNA concentration 31
Fig12、Light micrograph of particles isolated from goat mammary secretion with the cytospin certrifuge technique 34
Fig13、Light micrograph of particles isolated from goat mammary secretion with the cytospin certrifuge technique and treated with Wright''s stain 35
Fig14、Morphology of epithelial cell isolated from goat mammary secretion with the cytospin certifuge technique and stained with monoclonal anti-cytokeratin (human) peptide 7-FITC conjugate 36
Fig15、Light micrograph of particles isolated from goat mammary secretion on a week following dry off with the cytospin certrifuge technique and treated with Wright''s stain 37
Fig16、Electrophoretic analysis of DNA integrity in cell of mammary secretions of goats during different lactation stages 40
Fig17、Electrophoretic analysis of DNA integrity in mammary secretions of goats during different lactation stages 41
Fig18、Progressive change of superoxide dismutase (SOD) activity in non-cellular mammary secretion of individual goats during different lactation stages 44
Fig19、Progressive change of the glutathione peroxidase (GPx) activity in non-cellular mammary secretion of individual goats during different lactation stages 46
Fig20、Progressive change of the concentration of thiobarburic acid reactive substance (TBARS) in non-cellular mammary secretion of individual goats during different lactation stages 47
Fig21、Progressive change of the cellular superoxide dismutase (SOD) activity in mammary secretion of individual goats during different lactation stages 50
Fig22、Progressive change of the cellular glutathione peroxidase (GPx) activity in mammary secretion of individual goats during the different lactation stages 51
Fig23、Progressive change of the concentration of cellular thiobarburic acid reactive substance (TBARS) in mammary secretion of individual goats during the different lactation stages 53
表次
Table1、Morphological differences and similarities between apoptosis and necrosis 8
Table2、Biochmical differences and similarities between apoptosis and necrosis 8
Table3、Means of DMSCC (direct microscopic somatic cell count) and total cellular DNA, and percentages of DNA fragmentation in mammary secretions of goats during different lactation stages 38
Table4、Means activities of superoxide dismutase (SOD)、glutathione peroxidase (GPx) and thiobarburic acid reactive substances (TBARS) in non-cellular mammary secretion of goats during different lactation stages 43
Table5、Means activities of cellular superoxide dismutase (SOD)、glutathione peroxidase (GPx) and thiobarburic acid reactive substances (TBARS) in mammary secretion of goats during different lactation stages 49
Table6、Correlation coefficients among cellular activities of superoxide dismutase (SOD)、and glutathione peroxidase (GPx)、thiobarburic acid reactive substances (TBARS)、total cellular DNA、percentages of DNA fragmentation and means of DMSCC (direct microscopic somatic cell count) in mammary secretions of goats during different lactation stages 55
Table7、Correlation coefficients among cellular activities of superoxide dismutase (SOD)、and glutathione peroxidase (GPx)、thiobarburic acid reactive substances (TBARS) in non-cellular mammary secretion of goats VS. cellular SOD、GPx activity、TBARS、 total cellular DNA、percentages of DNA fragmentation and means of DMSCC (direct microscopic somatic cell count) in mammary secretions of goats during different lactation stages 56

參考文獻
李平南。1998。短期維生素E注射對熱季荷蘭牛抗氧化能力及泌乳功能隻影響。碩士論文。國立中興大學。台中市。
林峻暐。1999。Resveratrol,4-hexylresorcinol及trolox之抗氧化特性和對不同細胞DNA氧化傷害及抗氧化酵素活性影響之研究。碩士論文。國立中興大學。台中市。
Anne, S., Middleton, E. J. and R. E. Reisman. 1994. Vancomycin anaphylaxis and successful desensitization. Ann. Allergy 73:402-404.
Arends, M. J., R. G. Morris and A. H. Wyllie. 1990. Apoptosis：the role of the endonuclease. Am. J. Pathol. 136：593-608.
Atwood, C. S., M. Ikeda and B. K. Vonderhaar. 1995. Involution of mouse mammary glands in whole organ culture：a model for studying programmed cell death. Biochem. Biophys. Res. Commun. 207：860-867.
Aurand, L. W., N. H. Boone and G. G. Giddings. 1976. Superoxide and singlet oxygen in milk lipid peroxidation. J. Dairy Sci. 60：363-369.
Barr, P. J. and L. David. 1994. Apoptosis and its role in human disease. Bio/Technology 12：487-493.
Bay, B., Y. Lee, B. K. Tan and E. Ling. 1999. Lipid peroxidative stress and antioxidative enzymes in brains of milk-supplemented rats. Neurosci. Lett. 277：127-130.
Bendich, A. 1990. Antioxidant nutrients and immune functions introduction. In Bendich, A.（Ed.）. Antioxidant nutrients and immune functions. pp：l-12. Plenum Press. New York and London.
Benedetti, A., Comporti, M. and H. Esterbauer. 1980. Identification of 4-hydroxynonenal as a cytotoxic product orginating from the peroxidation of liver microsimal lipids. Biochim. Biophys. Acta. 620:281-296.
Brinboim, H. C. 1990. DNA strand breaks in human leukocytes induced by superoxide anion, hydrogen peroxide and tumor promoters are repaired slowly compared to breaks induced by ionizing radiation. Carcinogenesis. 7：1511-1517.
Buehring, G. C. 1990. Culture of mammary epithelial cells from bovine milk. J. Dairy Sci. 73：956-963.
Burton, K. 1956. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem. J. 63：315-323.
Cadenas, E. 1989.Biochemistry of oxygen toxicity. Annu. Rev. Biochem. 58:79-110.
Chen, H., Pellett, L. J., Andersen, H. J. and A. L. Tappel. 1993. Protection by vitamin E, selenium, and beta-carotene against oxidative damage in rat liver slices and homogenate. Free Radic. Biol. Med. 14:473-482.
Chow, C. K. 1992. Nutrition and cellular antioxidant defense. J. Chin. Nutr. Soc. 17：1-14.
Ciriolo, M. R., K. Fiskin and A. Martino. 1991. Age-releated changes in Cu, Zn superoxide dismutase, Se-dependent and —independent glutathione peroxidase and catalase activities in specific areas of rat brain. Mech. Ageing Dev. 61：287-297.
Cochrane, C. G. 1991. Mechanisms of oxidant injury of cell. Mol. Aspects Med. 12:137-147.
Comporti, M. 1987. Glutathione delepting agents and lipid peroxidation. Chem. Phys. Lipids 45:143-169.
Cristiano, F., J. B. Haan, R. C. Iannello and I. Kola. 1995. Changes in the levels of enzymes which modulate the antioxidant balance occur during aging and correlate with cellular damage. Mech. Ageing Dev. 80：93-105.
Dean, R. T. Thomas, S. M. and Garner, A. 1986. Free-radical-mediated fragmentation of monoamine oxidase in the mitochondrial membrane. Roles for lipid radical. Biochem. J. 240:489-494.
Deme, D., Virion, A., Hammou, N. A. and J. Pommier. 1985. NADPH-dependent generation of H2O2 in a thyroid particulate fraction requires Ca2+. FEBS Lett. 186:107-110.
Donnelly, J. K., K. M. McLellan, J. L. Walker and D. S. Robinson. 1988. Superoxide dismutase in foods：a review. Food Chemistry 33：243-270.
Dulin, A. M., M. J. Paape and B. T. Weinland. 1982. Cytospin centrifuge in differential counts of milk somatic cells. J. Dairy Sci. 65：1247-1251.
Dulin, A. M., M. J. Paape and W. P. Wergin. 1982. Differentiation and enumeration of somatic cells in goat milk. J. Food Prot. 45：435-439.
Dulin, A. M., M. J. Paape, W. D. Schultze and B. T. Weinland. 1983. Effect of Parity, stage of lactation, and intramammary infection on concentration of somatic cells and cytoplasmic particles in goat milk. J. Dairy Sci. 66：2426-2433.
Dunkley, W. L. and W. G. Jennings. 1951. A procedure for application of the thiobarbituric acid test to milk. Biochem. J. ：1064-1069.
Esterbauer, H., Benedetti, A., Lang, J., Fulceri, R., Fauler, G. and M. Comporti. 1986. Studies on the mechanism of formation of 4-hydroxynonenal druing microsomal lipid peroxidation. Biochim. Biophys. Acta. 876:154-166.
Esterbauer, H., Schauur, J. S. and H. Zollner. 1991. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehydes. Free Radic. Biol. Med. 11:81-128.
Floyd, R. A. 1990. Role of oxygen free radicals in carcinogenesis and brain ischemia. FASEB J. 4:2587-2597.
Guarente, L. 1999. Mutant mice live longer. Nature 402：243-245.
Halliwell, B. and S. Chirico. 1993. Lipid peroxidation: its mechanism, measure and significance. Am. J. Clin. Nutr. 57:715-725.
Halliwell, B. and J. M. C. Gutteridage. 1989. "Free Radical in Biology and Medicine", ed. by B. Halliwell and J. M. C. Gutteridge. Clarendon Press,Oxford.
Halliwell, B. and J. M. C. Gutteridge. 1984. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem. J. 219:1-4.
Halliwell, B., J. M. C. Gutteridge and C. E. Cross. 1992. Free radicals, antioxidants and human disease：where are we now？ J. Lab. Clin. Med. 199：598-620.
Heermeier, K., M. Benedict, M. Li, P. Furth, G. Nunez and L. Hennighausen. 1996. Bax and Bcl-xs are induced at the onset of apoptosis in involuting mammary epithelial cells. Mechanisms of development 56：197-207.
Hicks, C. L. 1980. Occurrence and consequence of superoxide dismutase in milk products：a review. J. Dairy Sci. 63：1199-1204.
Hicks, C. L., M. K. Dahl and T. Richardson. 1975. Superoxide dismutase in bovine milk. J. Dairy Sci. 58：796.
Hockenbery, D. M., Z. N. Oltvai, X. M. Yin, C. L. Milliman and S. J. Korsmeyer. 1993. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75：241-251.
Hogan, J. S., W. P. Weiss and K. L. Smith. 1993. Role of vitamin E and selenium in host defense against mastitis. J. Dairy Sci. 76：2795-2803.
Holst, B. D., W. L. Hurley and D. R. Nelson. 1987. Involution of the bovine mammary gland：histological and ultrastructural changes. J. Dairy Sci. 70：935-944.
Hu, Z., T. Ito, K. Y. C. Xie, H. Ozawa and M. Kawata. 1998. In vivo time course of morphological changes and DNA degradation during the degeneration of castration-induced apoptotic prostate cells. Cell Tissue Res. 294：153-160.
Hyslop, P. A., Hinshaw, D. B. and W. A. Halsey. 1988. Mechanisms of oxidant-mediated cell injury : the glycolytic and mitochondrial pathways of ADP phosphorylation are major intracellular targets inactivated by hydrogen peroxide. J. Biol. Chem. 263:1665-1675.
Imlay, J. A. and I. Fridovich. 1991. Assay of metabolic superoxide production in Escherichia coli. J. Biol. Chem. 266:6957-6965.
Kane, D. J., T. A. Sarafian, R. Anton, H. Hahn, E. B. Gralla, J. S. Valentine, T. Ord and D. E. Bredesen. 1995. Bcl-2 inhibition of neural death：decreased generation of reactive oxygen species. Science 262：1274-1276.
Kanner, J., German, J. B. and J. E. Kinsella. 1987. Initation of lipid peroxidation in biological systems. Crit. Rev. Food Sci. Nutr. 25:317-363.
Kerr, J. F. R., C. M. Winterford and B. V. Harmon. 1994. Morphological Criteria for Identifying Apoptosis. In：Celis, J. E.（Ed.）. Cell Biology. pp：319-329. Academic press. San Diego. CA.
Kerr, J. F. R., J. Searle. B. V. Harmon and C. J. Bishop. 1987. Apoptosis. In：Potten , C. S. （Ed.）.Perspectives on Mammalian Cell Death. pp. 93-128. Oxford university press. New York.
Kitchen, B. J. 1976. Enzymic methods for estimation of the somatic cell count in bovine milk. J. Dairy Res. 43：251-258.
Kroemer. G., P. Petit, N. Zamzami, J. Vayssiere and B. Mignotte. 1995. The biochemistry of programmed cell death. FASEB J. 9：1277-1287.
Lawrence, R. and R. Burk. 1976. Glutathione peroxidase activity in selenium deficient rat liver. Biochem. Biophys. Res. Commun. 71：952-958.
Liu, T. Y., Chen, C. L. and C. W. Chi. 1996. Oxidative damage to DNA induced by areca nut extract. Mutat. Res. 367:125-31.
Lizard, G., S. Gueldry, O. Sordet, S. Monier, A. Athias, C. Miguet, G. Bessede, S. Lemaire, E. Solary and P. Gambert. 1998. Glutathione is implied in the control of 7-ketocholesterol-induced apoptosis, which is associated with radical oxygen species production. FASEB J. 12：1651-1663.
Mailer, K., I. Macleod and W. Morris. 1991. Age related changes in anti-oxidative enzymes in cardiomyopathic hamster hearts. Mech. Ageing Dev. 59：37-45.
Marklund, S. and G. Marklund. 1974. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur. J. Biochem. 47：469-474.
Meier, B., Radeke, H., Shelle, S., Raspe, H. H., Resch, K. and G. G. Habermehl. 1990. Human fibroblasts release reactive oxygen species in response to treatment with synovial fluids from patients suffering from arthitis. Free Rad. Res. Commun. 8:149.
Mepham, T. B. 1987. Physiology of Lactation. Open University press. London.
Oberley, T. D., J. L. Schultz, N. Li and L. W. Oberley. 1995. Antioxidant enzyme levels as a function of growth state in cell culture. Free Radic. Biol. Med. 19：53-65.
Olinksi, R. T., Zastaway, J. Budzbon, J. Skokowski, W. and M. D. Zegarski. 1992. DNA modification in chromatin of human cancerous tissues. FEBS Lett. 309:193-198.
Paglia, D. E. and W. N. Valentine. 1967. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J. Lab. Clin. Med. 70：158-169.
Poli, G., Albano, E. and M. U. Dianzani. 1987. The role of lipid peroxidation in liver damage. Chem. Phys. Lipids 45:117-142.
Quarrie, L. H., C. V. P. Addey and C. J. Wilde. 1995. Apoptosis in lactating and involuting mouse mammary tissue demonstrated by nick-end DNA labeling. Cell Tissue Res. 281：413-419.
Ren, J., R. Zheng, Y. Shi, B. Gong and J. Li. 1998. Apoptosis, redifferentiation and arresting proliferation simultaneously triggered by oxidative stress in human hepatoma cells. Cell Biol. Int. 22：41-49.
Richards, D. M., Dean, R. T. and W. Jessup. 1988. Membrane proteins are critical targets in free radical mediated cytolysis. Biochim. Biophys. Acta946:281-288.
Rota, A. M., C. Gonzalo, P. L. Rodriguez, A. I. Rojas, L. Martin and J. J. Tovar. 1993. Somatic cell types in goats milk in relation to total cell count, stage and number of lactation. Small Rumin. Res. 12：89-98.
Shindo, Y. and T. Hashimoto. 1998. Ultraviolet B-induced cell death in four cutaneous cell lines exhibiting different enzymatic antioxidant defences：involvement of apoptosis. J. Dermatol. Sci. 17：140-150.
Siesjo, B. K., Agardh, C. D. and F. Bengtsson. 1989. Free radicals and brain damage. Cerebrovasc. Brain Metab. Rev. 1:165-211.
Simic, S. 1994. On the 100th anniversary of the Gynecology-Obstetrical Clinic in Sarajevo. Med.Arh. 48:191-196.
Slater, A. F. G., C. Stefan and S. Orrenius. 1995. The role of intracellular oxidants in apoptosis. Biochimica et Biophysica Acta 1271：59-62.
Steinbrecher, U. P. and P. H. Pritchard. 1989. Hydrolysis of phosphatidylcholine during LDL oxidation is mediated by platelet-avtivating factor acetyhydrolase. J. Lipid Res. 59:578-585.
Strange, R., F. Li, S. Saurer, A. Burkhardt and R. R. Friis. 1992. Apoptotic cell death and tissue remodeling during mouse mammary gland involution. Development 115：49-58.
Vuillaume, M. 1987. Reduced oxygen species, mutation, induction and cancer initiation. Mutat Res. 186:43-72.
Walker, N. I. R. E. Bennett and J. F. R. Kerr. 1989. Cell death by apoptosis during involution of the lactating breast in mice and rats. Am. J. Anat. 185：19-32.
Wei, T., C. Chen, J. Hou, B. Zhao, W. Xin and A. Mori. 1999. The antioxidant EPC-K1 attenuates NO-induced mitochondrial dysfunction, lipid peroxidation and apoptosis in cerebellar granule cells. Toxicology 134：117-126.
Wilde, C. J., L. H. Quarrie, E. Tonner and D. J. Flint. 1997. Mammary apoptosis. Livest. Prod. Sci. 50：29-37.
Wolfe, J. T., J. H. Pringle and G. M. Cohen. 1996. Assayys for the measurement of DNA fragmentation during apoptosis. In：Techniques in apoptosis. Portland press, London, P：51-70.
Wooding, F. B. P., M. Peaker and J. L. Linzell. 1970. Theories of milk secretion：evidence from the electron microscopic examination of milk. Nature 226：762-764.