臺灣博碩士論文加值系統

目前針對花生四烯酸（arachidonic acid，AA C20:4 ω6）的研究指出，AA在細胞中具有兩種截然不同的功能。一為增加氧化壓而導致細胞凋亡，另一方面則具有促使細胞增生、乃至癌化之現象。然而現今對AA在乳癌細胞上的作用並不清楚。因此本實驗之目的為：以人類乳腺癌細胞，MCF-7，為材料，探討AA是否會引發乳癌細胞進行凋亡。實驗方法為：利用2% FBS DMEM中補充不同濃度之AA處理六小時，檢測其影響。

結果發現：給予MCF-7細胞0、10、25、50、100、200 μM 的AA處理六小時後，可改變MCF-7細胞中各脂肪酸之比例。其中AA佔總脂肪酸的比例可由控制組（AA 0 μM）之7.4%上升至給予50 μM時的15％，但超過50 μM之後，AA在細胞中之比例則不再增加。若分別給予0、10、50、100 μM的AA處理六小時後，相較於控制組，AA的處理並不會改變MCF-7細胞的存活率。以流式細胞儀偵測給予0、10、50、100 μM的AA處理四、六、八、十六小時後的粒線體膜電位，發現MCF-7細胞之粒線體膜電位並未出現去極化現象。同時給予MCF-7細胞處理0、10、50、100 μM 的AA六小時後，與細胞凋亡相關之酵素：caspase-7與caspase-9均無顯著增加。

本實驗結果證明，在人類乳腺癌細胞上，給予AA處理六小時內並不會造成其存活率之下降，也不會影響細胞凋亡之兩項指標：粒線體膜電位去極化、以及procaspase-7、procaspase-9的活化。顯示AA對於人類乳腺癌細胞並無引發細胞凋亡的效果。

Arachidonic acid (20:4ω6, AA) may involve the cell proliferation or apoptosis shown in many studies. However, it is still unknown whether AA could induce apoptosis in MCF-7, a human breast cancer cell lines. The aim of this study was to investigate effect of AA on apoptosis in MCF-7. Cells was cultured in DMEM with 2% FBS for 42 hours, followed by AA supplementation for 6 hours.
It was found that the AA level in MCF-7 was increased from 7.4% of total fatty acid up to 15% with the increasing AA supplementation to 50 μM. However, the viability of MCF-7 was not changed compared with controls with 10, 50, and 100 μM AA supplementation. The mitochondrial membrane potential was not altered in MCF-7 supplemented with 10, 50, and 100 μM AA for 4, 6, 8 or 16 hours. The activation of procaspase-7 and procaspase-9 were not increased with the increasing AA treatment.
In conclusion, the increased AA level in MCF-7 did not change its viability, mitochondrial membrane potential, caspase-7 and caspase-9.

中文摘要 I
Abstract II
第壹章、緒論 1
一、研究動機 1
二、實驗假設 2
三、實驗設計 2
四、重要性 2
第貳章、文獻回顧 3
一、女性乳癌簡介 3
二、多元不飽和脂肪酸 4
三、花生四烯酸與其代謝產物 5
四、花生四烯酸與細胞增生、死亡之相關性 7
五、花生四烯酸與細胞凋亡之相關性 8
第參章、材料與方法 11
一、實驗材料 11
二、花生四烯酸儲備品之配製 11
三、脂肪酸萃取與甲基化反應 12
四、脂肪酸成分分析 13
（一）、氣相層析儀（Gas chromatography）簡介 13
（二）、操作流程 13
（三）、數據處理 14
五、細胞存活率檢測 15
六、粒線體膜電位偵測 15
（一）、流式細胞儀（Flow cytometry）簡介 15
（二）、螢光染劑DIOC6(3)與PI之製備 16
（三）、DIOC6(3)與PI雙染之原理 16
（四）、實驗流程 17
（五）、流式細胞儀之應用軟體與操作流程 18
（六）、數據處理 21
七、西方墨點法 21
（一）、實驗條件 21
（二）、蛋白質萃取 22
（三）、製作蛋白質標準曲線、測量樣本蛋白質濃度 23
（四）、SDS膠體電泳（SDS-PAGE）分析 23
（五）、SDS-PAGE流程 25
（六）、蛋白質轉印流程 26

（七）、抗體反應 27
（八）、暗房壓片與定量 29
八、統計方法 30
九、實驗器材、藥品一覽表 30
第肆章、實驗結果 33
一、脂肪酸成分分析 33
二、細胞存活率檢測 36
三、粒線體膜電位偵測 37
四、西方墨點法分析 41
第伍章、討論 43
一、實驗設計部分 43
（一）、實驗材料的選擇 43
（二）、AA的配製與儲存方法 43
（三）、培養條件 44
（四）、處理時間 45
二、實驗結果部分 45
（一）、AA在癌細胞與正常細胞間之比例差異 45
（二）、AA對細胞存活率的影響 47
（三）、AA對粒線體膜電位的影響 48
（四）、AA對caspase-7與caspase-9的影響 50
第陸章、結論 52
第柒章、文獻回顧 53

Antonsson, B., F. Conti, A. Ciavatta, S. Montessuit, S. Lewis, I. Martinou, L. Bernasconi, A. Bernard, J.J. Mermod, G. Mazzei, K. Maundrell, F. Gambale, R. Sadoul, and J.C. Martinou. 1997. Inhibition of Bax channel-forming activity by Bcl-2. Science. 277:370-2.
Basanez, G., A. Nechushtan, O. Drozhinin, A. Chanturiya, E. Choe, S. Tutt, K.A. Wood, Y. Hsu, J. Zimmerberg, and R.J. Youle. 1999. Bax, but not Bcl-xL, decreases the lifetime of planar phospholipid bilayer membranes at subnanomolar concentrations. Proc Natl Acad Sci U S A. 96:5492-7.
Bennett, F.C., and D.M. Ingram. 1990. Diet and female sex hormone concentrations: an intervention study for the type of fat consumed. Am J Clin Nutr. 52:808-12.
Borgeat, P., and P.H. Naccache. 1990. Biosynthesis and biological activity of leukotriene B4. Clin Biochem. 23:459-68.
Brooks, S.C., E.R. Locke, and H.D. Soule. 1973. Estrogen receptor in a human cell line (MCF-7) from breast carcinoma. J Biol Chem. 248:6251-3.
Buendia, B., A. Santa-Maria, and J.C. Courvalin. 1999. Caspase-dependent proteolysis of integral and peripheral proteins of nuclear membranes and nuclear pore complex proteins during apoptosis. J Cell Sci. 112 ( Pt 11):1743-53.
Cao, Y., A.T. Pearman, G.A. Zimmerman, T.M. McIntyre, and S.M. Prescott. 2000. Intracellular unesterified arachidonic acid signals apoptosis. Proc Natl Acad Sci U S A. 97:11280-5.
Cuendet, M., and J.M. Pezzuto. 2000. The role of cyclooxygenase and lipoxygenase in cancer chemoprevention. Drug Metabol Drug Interact. 17:109-57.
Daniel, P.T., T. Wieder, I. Sturm, and K. Schulze-Osthoff. 2001. The kiss of death: promises and failures of death receptors and ligands in cancer therapy. Leukemia. 15:1022-32.
Das, U.N. 2006. Essential fatty acids: biochemistry, physiology and pathology. Biotechnol J. 1:420-39.
Decordier, I., E. Cundari, and M. Kirsch-Volders. 2005. Influence of caspase activity on micronuclei detection: a possible role for caspase-3 in micronucleation. Mutagenesis. 20:173-9.
Desagher, S., and J.C. Martinou. 2000. Mitochondria as the central control point of apoptosis. Trends Cell Biol. 10:369-77.
Dymkowska, D., J. Szczepanowska, M.R. Wieckowski, and L. Wojtczak. 2006. Short-term and long-term effects of fatty acids in rat hepatoma AS-30D cells: the way to apoptosis. Biochim Biophys Acta. 1763:152-63.
Edwards, D.P., and W.L. McGuire. 1980. 17 alpha-Estradiol is a biologically active estrogen in human breast cancer cells in tissue culture. Endocrinology. 107:884-91.
Ferguson, H.A., P.M. Marietta, and C.L. Van Den Berg. 2003. UV-induced apoptosis is mediated independent of caspase-9 in MCF-7 cells: a model for cytochrome c resistance. J Biol Chem. 278:45793-800.
Finstad, H.S., M.C. Myhrstad, H. Heimli, J. Lomo, H.K. Blomhoff, S.O. Kolset, and C.A. Drevon. 1998. Multiplication and death-type of leukemia cell lines exposed to very long-chain polyunsaturated fatty acids. Leukemia. 12:921-9.
Funk, C.D. 2001. Prostaglandins and leukotrienes: advances in eicosanoid biology. Science. 294:1871-5.
Garrido, R., A. Malecki, B. Hennig, and M. Toborek. 2000. Nicotine attenuates arachidonic acid-induced neurotoxicity in cultured spinal cord neurons. Brain Res. 861:59-68.
Germain, M., E.B. Affar, D. D''Amours, V.M. Dixit, G.S. Salvesen, and G.G. Poirier. 1999. Cleavage of automodified poly(ADP-ribose) polymerase during apoptosis. Evidence for involvement of caspase-7. J Biol Chem. 274:28379-84.
Goetzl, E.J., S. An, and W.L. Smith. 1995. Specificity of expression and effects of eicosanoid mediators in normal physiology and human diseases. Faseb J. 9:1051-8.
Gottfried, E.L. 1967. Lipids of human leukocytes: relation to celltype. J Lipid Res. 8:321-7.
Green, D.R., and G. Kroemer. 2004. The pathophysiology of mitochondrial cell death. Science. 305:626-9.
Green, D.R., and J.C. Reed. 1998. Mitochondria and apoptosis. Science. 281:1309-12.
Haag, M. 2003. Essential fatty acids and the brain. Can J Psychiatry. 48:195-203.
Halestrap, A.P., S.J. Clarke, and S.A. Javadov. 2004. Mitochondrial permeability transition pore opening during myocardial reperfusion--a target for cardioprotection. Cardiovasc Res. 61:372-85.
Hamilton, J.A., S. Era, S.P. Bhamidipati, and R.G. Reed. 1991. Locations of the three primary binding sites for long-chain fatty acids on bovine serum albumin. Proc Natl Acad Sci U S A. 88:2051-4.
Haworth, R.A., and D.R. Hunter. 1979. The Ca2+-induced membrane transition in mitochondria. II. Nature of the Ca2+ trigger site. Arch Biochem Biophys. 195:460-7.
Henderson, W.R., Jr. 1994. The role of leukotrienes in inflammation. Ann Intern Med. 121:684-97.
Hengartner, M.O. 2000. The biochemistry of apoptosis. Nature. 407:770-6.
Hofmanova, J., A. Vaculova, and A. Kozubik. 2005a. Polyunsaturated fatty acids sensitize human colon adenocarcinoma HT-29 cells to death receptor-mediated apoptosis. Cancer Lett. 218:33-41.
Hofmanova, J., A. Vaculova, A. Lojek, and A. Kozubik. 2005b. Interaction of polyunsaturated fatty acids and sodium butyrate during apoptosis in HT-29 human colon adenocarcinoma cells. Eur J Nutr. 44:40-51.
Holt, J.T., M.E. Thompson, C. Szabo, C. Robinson-Benion, C.L. Arteaga, M.C. King, and R.A. Jensen. 1996. Growth retardation and tumour inhibition by BRCA1. Nat Genet. 12:298-302.
Horwitz, K.B., M.E. Costlow, and W.L. McGuire. 1975. MCF-7; a human breast cancer cell line with estrogen, androgen, progesterone, and glucocorticoid receptors. Steroids. 26:785-95.
Janssen-Timmen, U., I. Tomic, E. Specht, U. Beilecke, and A.J. Habenicht. 1994. The arachidonic acid cascade, eicosanoids, and signal transduction. Ann N Y Acad Sci. 733:325-34.
Jemal, A., R. Siegel, E. Ward, T. Murray, J. Xu, and M.J. Thun. 2007. Cancer statistics, 2007. CA Cancer J Clin. 57:43-66.
Jensen, E.V., E.R. Desombre, D.J. Hurst, T. Kawashima, and P.W. Jungblut. 1967. Estrogen-receptor interactions in target tissues. Arch Anat Microsc Morphol Exp. 56:547-69.
Kachhap, S.K., P. Dange, and S. Nath Ghosh. 2000. Effect of omega-6 polyunsaturated fatty acid (linoleic acid) on BRCA1 gene expression in MCF-7 cell line. Cancer Lett. 154:115-20.
Kerr, J.F., A.H. Wyllie, and A.R. Currie. 1972. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 26:239-57.
Klock, J.C., and J.K. Pieprzyk. 1979. Cholesterol, phospholipids, and fatty acids of normal immature neutrophils: comparison with acute myeloblastic leukemia cells and normal neutrophils. J Lipid Res. 20:908-11.
Kothakota, S., T. Azuma, C. Reinhard, A. Klippel, J. Tang, K. Chu, T.J. McGarry, M.W. Kirschner, K. Koths, D.J. Kwiatkowski, and L.T. Williams. 1997. Caspase-3-generated fragment of gelsolin: effector of morphological change in apoptosis. Science. 278:294-8.
Kyprianou, N., H.F. English, N.E. Davidson, and J.T. Isaacs. 1991. Programmed cell death during regression of the MCF-7 human breast cancer following estrogen ablation. Cancer Res. 51:162-6.
Lewis, C.J., and E.A. Yetley. 1999. Health claims and observational human data: relation between dietary fat and cancer. Am J Clin Nutr. 69:1357S-1364S.
Liang, Y., C. Yan, and N.F. Schor. 2001. Apoptosis in the absence of caspase 3. Oncogene. 20:6570-8.
Liebens, F.P., B. Carly, A. Pastijn, and S. Rozenberg. 2007. Management of BRCA1/2 associated breast cancer: A systematic qualitative review of the state of knowledge in 2006. European Journal of Cancer. 43:238-257.
Madhavi, N., U.N. Das, P.S. Prabha, G.S. Kumar, R. Koratkar, and P.S. Sagar. 1994. Suppression of human T-cell growth in vitro by cis-unsaturated fatty acids: relationship to free radicals and lipid peroxidation. Prostaglandins Leukot Essent Fatty Acids. 51:33-40.
McEntee, M.F., and J. Whelan. 2002. Dietary polyunsaturated fatty acids and colorectal neoplasia. Biomed Pharmacother. 56:380-7.
McPherson, K., C.M. Steel, and J.M. Dixon. 2000. ABC of breast diseases. Breast cancer-epidemiology, risk factors, and genetics. Bmj. 321:624-8.
Pepe, S. 2005. Effect of dietary polyunsaturated fatty acids on age-related changes in cardiac mitochondrial membranes. Exp Gerontol. 40:751-8.
Petit, P., N. Glab, D. Marie, H. Kieffer, and P. Metezeau. 1996. Discrimination of respiratory dysfunction in yeast mutants by confocal microscopy, image, and flow cytometry. Cytometry. 23:28-38.
Petronilli, V., D. Penzo, L. Scorrano, P. Bernardi, and F. Di Lisa. 2001. The mitochondrial permeability transition, release of cytochrome c and cell death. Correlation with the duration of pore openings in situ. J Biol Chem. 276:12030-4.
Pompeia, C., J.J. Freitas, J.S. Kim, S.B. Zyngier, and R. Curi. 2002. Arachidonic acid cytotoxicity in leukocytes: implications of oxidative stress and eicosanoid synthesis. Biol Cell. 94:251-65.
Pompeia, C., T. Lima, and R. Curi. 2003. Arachidonic acid cytotoxicity: can arachidonic acid be a physiological mediator of cell death? Cell Biochem Funct. 21:97-104.
Rao, L., D. Perez, and E. White. 1996. Lamin proteolysis facilitates nuclear events during apoptosis. J Cell Biol. 135:1441-55.
Rose, D.P. 1993. Diet, hormones, and cancer. Annu Rev Public Health. 14:1-17.
Schreiber, V., F. Dantzer, J.C. Ame, and G. de Murcia. 2006. Poly(ADP-ribose): novel functions for an old molecule. Nat Rev Mol Cell Biol. 7:517-28.
Scorrano, L., D. Penzo, V. Petronilli, F. Pagano, and P. Bernardi. 2001. Arachidonic acid causes cell death through the mitochondrial permeability transition. Implications for tumor necrosis factor-alpha aopototic signaling. J Biol Chem. 276:12035-40.
Semenov, D.V., P.A. Aronov, E.V. Kuligina, M.O. Potapenko, and V.A. Richter. 2004. Oligonucleosome DNA fragmentation of caspase 3 deficient MCF-7 cells in palmitate-induced apoptosis. Nucleosides Nucleotides Nucleic Acids. 23:831-6.
Shimizu, S., M. Narita, and Y. Tsujimoto. 1999. Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC. Nature. 399:483-7.
Shiose, A., and H. Sumimoto. 2000. Arachidonic acid and phosphorylation synergistically induce a conformational change of p47phox to activate the phagocyte NADPH oxidase. J Biol Chem. 275:13793-801.
Singh, B., and A. Lucci. 2002. Role of cyclooxygenase-2 in breast cancer. J Surg Res. 108:173-9.
Sivaprasad, U., E. Shankar, and A. Basu. 2007. Downregulation of Bid is associated with PKCepsilon-mediated TRAIL resistance. Cell Death Differ. 14:851-60.
Surette, M.E., J.D. Winkler, A.N. Fonteh, and F.H. Chilton. 1996. Relationship between arachidonate--phospholipid remodeling and apoptosis. Biochemistry. 35:9187-96.
Toraason, M., H. Wey, M. Woolery, P. Plews, and P. Hoffmann. 1995. Arachidonic acid supplementation enhances hydrogen peroxide induced oxidative injury of neonatal rat cardiac myocytes. Cardiovascular Research. 29:624-628.
Wenzel, D.C., and T.W. Hale. 1978. Toxicity of free fatty acids for cultured rat heart muscle and endothelioid cells. II. Unsaturated long-chain fatty acids. Toxicology. 11:119-25.
Wilson, H.A., B.E. Seligmann, and T.M. Chused. 1985. Voltage-sensitive cyanine dye fluorescence signals in lymphocytes: plasma membrane and mitochondrial components. J Cell Physiol. 125:61-71.
Wilson, J.W., A.E. Wakeling, I.D. Morris, J.A. Hickman, and C. Dive. 1995. MCF-7 human mammary adenocarcinoma cell death in vitro in response to hormone-withdrawal and DNA damage. Int J Cancer. 61:502-8.
Wynder, E.L., D.P. Rose, and L.A. Cohen. 1986. Diet and breast cancer in causation and therapy. Cancer. 58:1804-13.
Zoratti, M., and I. Szabo. 1995. The mitochondrial permeability transition. Biochim Biophys Acta. 1241:139-76.
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行政院衛生署民國九十五年死因統計資料
駱中郁, 民國94年, 探討紅花籽油攝取在促進荷爾蒙依賴型乳癌之作用機制及乳癌生成之關鍵時期生理學研究所,臺灣大學.