臺灣博碩士論文加值系統

鼻咽癌，俗稱「中國癌」，好發於華南地區。自然殺手細胞可以藉由perforin與granzyme B調控來毒殺癌細胞（細胞壞死與凋亡），然而血小板會壓抑自然殺手細胞毒殺癌細胞的能力。而且，不同的運動強度會影響不同程度的免疫反應與血小板活性。本研究是要探討短期運動如何影響血小板干擾自然殺手細胞毒殺鼻咽癌細胞的能力與其機制。30位健康靜態生活男性參與本實驗，分別進行中度、劇烈、中度－劇烈（60%, 100%, 60%－休息－100%最大攝氧量）固定式腳踏車運動，運動前後各抽取30毫升血液，用來偵測白血球、血小板數目變化；萃取出自然殺手細胞，觀察細胞內perforin與granzyme B含量；在有或無血小板的情況下，探討自然殺手細胞對鼻咽癌細胞的毒殺力（細胞壞死與凋亡）、caspase 3, 6和9活化，以及DNA斷裂的情形。結果顯示（1）劇烈運動增加自然殺手細胞數目與提升活性（perforin與granzyme B），同時提高血小板濃度；雖然劇烈運動提升自然殺手細胞毒殺鼻咽癌細胞的能力，但是同時也增加血小板的活性，進而阻礙自然殺手細胞毒殺力。（2）中度運動對自然殺手細胞數目、活性、血小板干擾自然殺手細胞與鼻咽癌細胞的結合、與鼻咽癌細胞之caspase活化情形，皆沒有顯著影響。並且血小板並不會影響自然殺手細胞毒殺鼻咽癌細胞的作用。（3）中度－劇烈運動提升自然殺手細胞數目與perforin與granzyme B含量；而且減少血小板干擾自然殺手細胞與鼻咽癌細胞的結合力，增加鼻咽癌細胞凋亡。本研究結果在臨床應用上提供一個新思維：中度－劇烈運動能增加自然殺手細胞數量與提升毒殺癌細胞能力，亦可降低血小板干擾自然殺手細胞的毒殺作用。

Background and Purpose: Nasopharyngeal carcinoma (NPC) is a common cancer in South China. Natural killer (NK) cells can induce the death of carcinoma cell by employing the perforin/granzyme-mediated cytotoxicity (necrosis and apoptosis), whereas platelets attenuate the ability of NK cells to destroy carcinoma cells. Exercise is associated with intensity-dependent immune response and platelet reactivity. This study was designed to investigate how short-term exercise affects platelet-impeded cytotoxicity of NK cell to NPC cells and its underlying mechanisms. Methods and Results: Thirty healthy sedentary men performed on three occasions, moderate exercise (ME, 60%VO2Max for 40 min), severe exercise (SE, up to VO2Max) and SE preceded by ME (MSE). Blood samples were collected before and immediately after exercise. Leukocyte and platelet counts, intracellular perforin and granzyme B contents in NK cells, cytotoxicity of NK cell to NPC cells, caspase-3, -8 and -9 activation as well as PS exposure (phosphatidylserine exposure) and DNA fragmentation of NPC cells were determined. The results demonstrated that (1) SE increased NK cell and platelet concentration in blood, and was accompanied by an elevation in perforin and granzym B levels of NK cells; although cytotoxicity of NK cell to NPC cells was enhanced by SE, co-incubating NK cells with platelets attenuated the ability of tumor PSexposure and DNA fragmentation following this exercise. (2) ME did not increase NK cell number and perforin and granzyme B contents, NK-NPC interaction as well as PS exposure and DNA fragmentation of NPC cells. (3) MSE increased NK cell, perforin and granzyme B. Moreover, with platelet, NK cell-NPC cell interaction enhanced contrarily and NPC cell apoptosis increased. Conclusions: SE increases the number of NK cell, and their perforin and granzyme B contents, NPC cell apoptosis, but simultaneously increases platelet counts and augments the extent of platelet-impeded cytotoxicity of NK cells to NPC cells. Conversely, ME influences neither NK-NPC interaction nor the functional behavior of NK/platelet with regard to NPC cells. MSE increases NK cell function and suppressed platelet-impeded cytotoxicity of NK cells to NPC cells. Therefore, NPC cell apoptosis increases. Clinical Relevance: The findings provide new insight into the benefit and the risk of exercise modulating survival and death of the tumor in patients with NPC.

指導教授推薦書
口試委員會審定書
授權書 iii
誌謝 iv
中文摘要 v
英文摘要 vii
中英名辭解釋 ix
目錄 xii
圖表目錄 xiv
第一章 緒論 1
第一節 研究背景及目的 1
第二節 研究假設 2
第二章 文獻回顧 3
第一節 鼻咽癌 3
第二節 運動對癌症的影響 9
第三節 運動劑量對免疫反應影響 10
第四節 自然殺手細胞 10
第五節 細胞死亡 13
第六節 自然殺手細胞造成癌細胞凋亡的途徑 15
第七節 運動劑量對自然殺手細胞毒殺力的影響 17
第八節 運動劑量對血小板活性的影響 17
第九節 癌細胞、自然殺手細胞與血小板之三角關係 18
第三章 實驗設計 20
第一節 研究設計 20
第二節 實驗材料 21
第三節 實驗方法 24
鼻咽癌細胞培養 26
血小板的置備 26
自然殺手細胞萃取 27
自然殺手細胞內perforin與granzyme B的表現 27
鼻咽癌細胞凋亡測定 28
自然殺手細胞與鼻咽癌細胞結合力測定 29
鼻咽癌細胞凋亡路徑（caspase-3, 8, 9）測定 30
鼻咽癌細胞DNA斷裂（DNA fragmentation）測定 30
電子細胞基質阻抗判斷分析（Electric cell-substrate impedance sensing (ECIS)） 31
數據分析（statistical analysis） 33
第四章 結果 34
第一節 受試者基本資料、身體狀況和活動情形調查 34
第二節 不同運動強度對血球數目數據的變化 34
第三節 不同運動強度對自然殺手細胞數量的影響 35
第四節 不同運動劑量對perforin與granzyme B數量的影響 35
第五節 不同運動強度對自然殺手細胞與癌細胞結合能力的影響 35
第六節 不同運動強度對鼻咽癌癌細胞caspase活化情形 36
第七節 不同運動強度對鼻咽癌癌細胞早期凋亡（PS外翻）的影響 36
第八節 不同運動強度影響鼻咽癌癌細胞凋亡情形（DNA斷裂） 37
第九節 不同運動強度對鼻咽癌癌細胞壞死的影響 38
第十節 不同運動強度影響細胞阻抗 38
第五章 討論 39
第一節 劇烈與中度－劇烈運動顯著增加自然殺手細胞數量與活性 39
第二節 血小板干擾自然殺手細胞與鼻咽癌細胞的毒殺作用 42
第六章 結論 46
圖表 48
參考文獻 77
附錄 90
附件一 受試者同意書 90
附件二 身體狀況與活動情形調查表 91
附件三 人體試驗倫理委員會同意臨床試驗證明書 92

中文參考文獻
1.李家鳳, 徐茂銘. 鼻咽癌. 當代醫學. 2002;29(5), 353-356.
2.許志宏. 鼻咽癌. 臨床醫學. 2002;50(2), 102-104.
3.江瑞坤, 高以信, 王美華, 劉玟娟, 蔡秀美, 黃子芸, 劉鎮嘉.鼻咽癌末期病人之照護：一個案報告. 安寧療護. 2005;10(1), 75-82
4.黃同村, 陳培榕, 徐莉萍東台灣鼻咽癌之治療經驗. 慈濟醫學. 2004;16, 159-165.
5.黃永任. 運動與自然殺手細胞. 中華體育. 1995;9 (2), 73-79.
6.黃森芳. 運動對人體免疫系統中自然殺手細胞功能之影響. 國民體育季刊. 1998;27(1), 32-45
7.林學宜, 林培元. 自然殺手細胞與運動關係之探討. 2000;48, 128-132
8.許福助, 「以流式細胞技術評估人類周邊血液白血球之免疫功能」, 臺北醫學大學, 碩士論文, 民國91年.
9.邱亦涵, 「高強度運動誘發氧化壓力對白血球凋亡之研究」, 台北巿立體育學院, 碩士論文, 民國91年.

英文參考文獻
Adrain C, Martin SJ. The mitochondrial apoptosome: A killer unleashed by the cytochrome seas. Trends in Biochemical Sciences. 2001;26(6):390-397.
Ameisen JC, Estaquier J, Idziorek T, De Bels F. The relevance of apoptosis to AIDS pathogenesis. Trends in Cell Biology. 1995;5(1):27-32.
Arndt S, Seebach J, Psathaki K, Galla HJ, Wegener J. Bioelectrical impedance assay to monitor changes in cell shape during apoptosis. Biosensors and Bioelectronics. 2004;19(6):583-594.
Aubry JP, Blaecke A, Lecoanet-Henchoz S, et al. Annexin V used for measuring apoptosis in the early events of cellular cytotoxicity. Cytometry. 1999;37(3):197-204.
Bauer S, Groh V, Wu J, et al. Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA. Science. Jul 30 1999;285(5428):727-729.
Blair SN. 1993 C.H. McCloy Research Lecture: physical activity, physical fitness, and health. Research Quarterly for Exercise and Sport. 1993;64(4):365-376.
Bloom ET, Babbitt JT. Prostaglandin E2, monocyte adherence and interleukin-1 in the regulation of human natural killer cell activity by monocytes. Natural Immunity and Cell Growth Regulation. 1990;9(1):36-48.
Brittenden J, Heys SD, Ross J, Eremin O. Natural killer cells and cancer. Cancer. 1996;77(7):1226-1243.
Bursch W, Oberhammer F, Schulte-Hermann R. Cell death by apoptosis and its protective role against disease. Trends in Pharmacological Sciences. 1992;13(6):245-251.
Chan ATC, Teo PML, Johnson PJ. Nasopharyngeal carcinoma. Annals of Oncology. 2002;13(7):1007-1015.
Chiappelli F, Yamashita N, Faisal M, et al. Differential effect of beta-endorphin on three human cytotoxic cell populations. International Journal of Immunopharmacology. 1991;13(2-3):291-297.
Chiorean EG, Miller JS. The biology of natural killer cells and implications for therapy of human disease. Journal of Hematotherapy and Stem Cell Research. 2001;10(4):451-463.
Dimeo F. Exercise for cancer patients: A new challenge in sports medicine. Western Journal of Medicine. 2000;173(4):272-273.
DiPenta JM, Green-Johnson J, Murphy RJL. Natural killer cells and exercise training in the elderly: A review. Canadian Journal of Applied Physiology. 2004;29(4):419-443.
Dohi K, Mastro AM, Miles MP, et al. Lymphocyte proliferation in response to acute heavy resistance exercise in women: influence of muscle strength and total work. Eur J Appl Physiol. Aug 2001;85(3-4):367-373.
Earnshaw WC, Martins LM, Kaufmann SH. Mammalian caspases: Structure, activation, substrates, and functions during apoptosis. Annual Review of Biochemistry. 1999;68:383-424.
Evans WJ, Meredith CN, Cannon JG. Metabolic changes following eccentric exercise in trained and untrained men. Journal of Applied Physiology. 1986;61(5):1864-1868.
Fadok VA, Laszlo DJ, Noble PW, Weinstein L, Riches DWH, Henson PM. Particle digestibility is required for induction of the phosphatidylserine recognition mechanism used by murine macrophages to phagocytose apoptotic cells. Journal of Immunology. 1993;151(8):4274-4285.
Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, Henson PM. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. Journal of Immunology. 1992;148(7):2207-2216.
Fiatarone MA, Morley JE, Bloom ET, Benton D, Makinodan T, Solomon GF. Endogenous opioids and the exercise-induced augmentation of natural killer cell activity. Journal of Laboratory and Clinical Medicine. 1988;112(5):544-552.
Gabriel H, Schwarz L, Born P, Kindermann W. Differential mobilization of leucocyte and lymphocyte subpopulations into the circulation during endurance exercise. European Journal of Applied Physiology and Occupational Physiology. 1992;65(6):529-534.
Gabriel H, Schwarz L, Steffens G, Kindermann W. Immunoregulatory hormones, circulating leucocyte and lymphocyte subpopulations before and after endurance exercise of different intensities. International Journal of Sports Medicine. 1992;13(5):359-366.
Gannon GA, Rhind SG, Suzui M, et al. beta-Endorphin and natural killer cell cytolytic activity during prolonged exercise. is there a connection? Am J Physiol. Dec 1998;275(6 Pt 2):R1725-1734.
Gasic GJ. Role of plasma, platelets, and endothelial cells in tumor metastasis. Cancer and Metastasis Reviews. 1984;3(2):99-114.
Golstein P, Ojcius DM, Young DE. Cell death mechanisms and the immune system. Immunological Reviews. 1991(121):29-65.
Hoffman SA, Paschkis KE, Debias DA, Cantarow A, Williams TL. The influence of exercise on the growth of transplanted rat tumors. Cancer Res. Jun 1962;22:597-599.
Hoffman-Goetz L. Exercise, natural immunity, and tumor metastasis. Medicine and Science in Sports and Exercise. 1994;26(2):157-163.
Houchins JP, Yabe T, McSherry C, Bach FH. DNA sequence analysis of NKG2, a family of related cDNA clones encoding type II integral membrane proteins on human natural killer cells. Journal of Experimental Medicine. 1991;173(4):1017-1020.
Jamieson AM, Diefenbach A, McMahon CW, Xiong N, Carlyle JR, Raulet DH. The role of the NKG2D immunoreceptor in immune cell activation and natural killing. Immunity. Jul 2002;17(1):19-29.
Janeway CA. Natural killer cells: a primitive immune system. Nature. 1989;341(6238):108.
Kappel M, Tvede N, Galbo H, et al. Evidence that the effect of physical exercise on NK cell activity is mediated by epinephrine. Journal of Applied Physiology. 1991;70(6):2530-2534.
Kärre K. NK cells, MHC class I molecules and the missing self. Scandinavian Journal of Immunology. 2002;55(3):221-228.
Keast D, Cameron K, Morton AR. Exercise and the immune response. Sports Medicine. 1988;5(4):248-267.
Kerr JF, Gobe GC, Winterford CM, Harmon BV. Anatomical methods in cell death. Methods in cell biology. 1995;46:1-27.
Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. British Journal of Cancer. 1972;26(4):239-257.
Klarlund Pedersen B. Modulation of natural killer cell activity in patients with immune-inflammatory diseases. Danish Medical Bulletin. 1988;35(4):315-322.
Landmann R. Beta-adrenergic receptors in human leukocyte subpopulations. European Journal of Clinical Investigation, Supplement. 1992;22(1):30-36.
Lewicki R, Tchorzewski H, Majewska E, Nowak Z, Baj Z. Effect of maximal physical exercise on T-lymphocyte subpopulations and on interleukin 1 (IL 1) and interleukin 2 (IL 2) production in vitro. International Journal of Sports Medicine. 1988;9(2):114-117.
Long EO, Rajagopalan S. Stress signals activate natural killer cells. Journal of Experimental Medicine. 2002;196(11):1399-1402.
Luger TA, Krutmann J, Kirnbauer R, et al. IFN-β2/IL-6 augments the activity of human natural killer cells. Journal of Immunology. 1989;143(4):1206-1209.
Maghazachi AA, Al-Aoukaty A. Chemokines activate natural killer cells through heterotrimeric G- proteins: Implications for the treatment of AIDS and cancer. FASEB Journal. 1998;12(11):913-924.
McNerlan SE, Rea IM, Alexander HD, Morris TC. Changes in natural killer cells, the CD57CD8 subset, and related cytokines in healthy aging. J Clin Immunol. Jan 1998;18(1):31-38.
Miles MP, Mackinnon LT, Grove DS, et al. The relationship of natural killer cell counts, perforin mRNA and CD2 expression to post-exercise natural killer cell activity in humans. Acta Physiol Scand. Apr 2002;174(4):317-325.
Miller JS. Biology of natural killer cells in cancer and infection. Cancer Investigation. 2002;20(3):405-419.
Moore C, Tittle PW. Muscle activity, body fat, and induced rat mammary tumors. Surgery. 1973;73(3):329-332.
Mould RF, Tai THP. Nasopharyngeal carcinoma: Treatments and outcomes in the 20th century. British Journal of Radiology. 2002;75(892):307-339.
Moyna NM, Acker GR, Weber KM, et al. Exercise-induced alterations in natural killer cell number and function. Eur J Appl Physiol Occup Physiol. 1996;74(3):227-233.
Na YM, Kim MY, Kim YK, Ha YR, Yoon DS. Exercise therapy effect on natural killer cell cytotoxic activity in stomach cancer patients after curative surgery. Archives of Physical Medicine and Rehabilitation. 2000;81(6):777-779.
Nieman DC. Exercise, infection, and immunity. International Journal of Sports Medicine. 1994;15(SUPPL. 3):S131-S141.
Nieswandt B, Hafner M, Echtenacher B, Männel DN. Lysis of tumor cells by natural killer cells in mice is impeded by platelets. Cancer Research. 1999;59(6):1295-1300.
Oldervoll LM, Kaasa S, Hjermstad MJ, Lund JA, Loge JH. Physical exercise results in the improved subjective well-being of a few or is effective rehabilitation for all cancer patients? European Journal of Cancer. 2004;40(7):951-962.
Palumbo JS, Talmage KE, Massari JV, et al. Platelets and fibrin(ogen) increase metastatic potential by impeding natural killer cell-mediated elimination of tumor cells. Blood. 2005;105(1):178-185.
Pedersen BK, Tvede N, Christensen LD, Klarlund K, Kragbak S, Halkjr-Kristensen J. Natural killer cell activity in peripheral blood of highly trained and untrained persons. International Journal of Sports Medicine. 1989;10(2):129-131.
Pedersen BK, Tvede N, Hansen FR, et al. Modulation of natural killer cell activity in peripheral blood by physical exercise. Scandinavian Journal of Immunology. 1988;27(6):673-678.
Pedersen BK, Tvede N, Klarlund K, et al. Indomethacin in vitro and in vivo abolishes post-exercise suppression of natural killer cell activity in peripheral blood. Int J Sports Med. Apr 1990;11(2):127-131.
Raff MC, Barres BA, Burne JF, Coles HS, Ishizaki Y, Jacobson MD. Programmed cell death and the control of cell survival: Lessons from the nervous system. Science. 1993;262(5134):695-700.
Rhind SG, Shek PN, Shinkai S, Shephard RJ. Effects of moderate endurance exercise and training on in vitro lymphocyte proliferation, interleukin-2 (IL-2) production, and IL-2 receptor expression. Eur J Appl Physiol Occup Physiol. 1996;74(4):348-360.
Rusch HP, Kline BE. The effect of exercise on the growth of a mouse tumor. Cancer research. 1944;4:116-118.
Schneider CM, Dennehy CA, Roozeboom M, Carter SD. A model program: Exercise intervention for cancer rehabilitation. Integrative Cancer Therapies. 2002;1(1):76-82.
Shounan Y, Feng X, O'Connell PJ. Apoptosis detection by annexin V binding: A novel method for the quantitation of cell-mediated cytotoxicity. Journal of Immunological Methods. 1998;217(1-2):61-70.
Smyth MJ, Cretney E, Kelly JM, et al. Activation of NK cell cytotoxicity. Molecular Immunology. 2005;42(4 SPEC. ISS.):501-510.
Spits H, Lanier LL, Phillips JH. Development of human T and natural killer cells. Blood. 1995;85(10):2654-2670.
Strasner A, Davis JM, Kohut ML, Pate RR, Ghaffar A, Mayer E. Effects of exercise intensity on natural killer cell activity in women. International Journal of Sports Medicine. 1997;18(1):56-61.
Takahara K, Miura Y, Kouzuma R, Yasumasu T, Nakamura T, Nakashima Y. Physical training augments plasma catecholamines and natural killer cell activity. Journal of UOEH. 1999;21(4):277-287.
Tan EM. Autoimmunity and apoptosis. Journal of Experimental Medicine. 1994;179(4):1083-1086.
Trapani JA, Smyth MJ. Functional significance of the perforin/granzyme cell death pathway. Nature Reviews Immunology. 2002;2(10):735-747.
Tvede N, Kappel M, Klarhund K, et al. Evidence that the effect of bicycle exercise on blood mononuclear cell proliferative responses and subsets is mediated by epinephrine. International Journal of Sports Medicine. 1994;15(2):100-104.
Tvede N, Steensberg J, Baslund B, Halkjaer-Kristensen J, Pedersen BK. Cellular immunity in highly trained elite racing cyclists during periods of training with high and low intensity. Scandinavian Journal of Medicine and Science in Sports. 1991;1(3):163-166.
Vetvicka V, Thornton BP, Wieman TJ, Ross GD. Targeting of Natural Killer Cells to Mammary Carcinoma via Naturally Occurring Tumor Cell-Bound iC3b and β-Glucan-Primed CR3 (CD11b/CD18). Journal of Immunology. 1997;159(2):599-605.
Voth R, Rossol S, Gallati H, et al. In vivo and in vitro induction of natural killer cells by cloned human tumor necrosis factor. Cancer Immunology Immunotherapy. 1988;27(2):128-132.
Wang JS, Cheng LJ. Effect of strenuous, acute exercise on α2-adrenergic agonist- potentiated platelet activation. Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19(6):1559-1565.
Wang JS, Jen CJ, Chen HI. Effects of chronic exercise and deconditioning on platelet function in women. Journal of Applied Physiology. 1997;83(6):2080-2085.
Wang JS, Jen CJ, Kung HC, Lin LJ, Hsiue TR, Chen HI. Different effects of strenuous exercise and moderate exercise on platelet function in men. Circulation. 1994;90(6):2877-2885.
Wang JS, Jen CJ, Lee HL, Chen HI. Effects of short-term exercise on female platelet function during different phases of the menstrual cycle. Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17(9):1682-1686.
Welsh RM, Brubaker JO, Vargas-Cortes M, O'Donnell CL. Natural killer (NK) cell response to virus infections in mice with severe combined immunodeficiency. The stimulation of NK cells and the NK cell-dependent control of virus infections occur independently of T and B cell function. Journal of Experimental Medicine. 1991;173(5):1053-1063.
Whiteside TL, Herberman RB. The role of natural killer cells in immune surveillance of cancer. Current Opinion in Immunology. 1995;7(5):704-710.
Williams GT. Role of apoptosis in the immune system. Biochemistry and cell biology = Biochimie et biologie cellulaire. 1994;72(11-12):447-450.
Woods JA, Davis JM, Smith JA, Nieman DC. Exercise and cellular innate immune function. Medicine and Science in Sports and Exercise. 1999;31(1):57-66.
Wyllie AH, Beattie GJ, Hargreaves AD. Chromatin changes in apoptosis. Histochemical Journal. 1981;13(4):681-692.
Wyllie AH, Morris RG. Hormone-induced cell death. Purification and properties of thymocytes undergoing apoptosis after glucocorticoid treatment. American Journal of Pathology. 1982;109(1):78-87.