臺灣博碩士論文加值系統

Sevoflurane是一種屬於普遍使用的吸入性麻醉劑。有幾個研究證實，在有心臟疾病下使用sevoflurane進行麻醉，會產生活性氧物質（reactive oxygen species），然而sevoflurane也可以直接觸發peroxynitrite的形成。而且就目前為止sevoflurane對於人類多型態核白血球（human polymorphonuclear neutrophils, PMN）是否會產生傷害並不清楚。本論文中，證實sevoflurane處理一小時會增加細胞內過氧化氫（H2O2）或過氧化物（peroxide）、超氧陰離子及一氧化氮（NO），並會造成細胞內的麩胱甘肽（gultathione, GSH）的消耗。過氧化氫酶（catalase）、甘露蜜醇（mannitol）、 dexamethasone、乙醯半胱胺酸（N-acetylcysteine , NAC）和水溶性維他命E（trolox）會抑制sevoflurane所誘發的過氧化氫（intracellular H2O2）或過氧化物（peroxide），但是超氧化物歧化酶（superoxide dismutase, SOD）則不會。就在上述結果發現過氧化氫酶（catalase）其會抑制sevoflurane所誘發的過氧化氫（H2O2）或過氧化物（peroxide）效果最好。所以當細胞處理sevoflurane後主要產生的活性氧物質是過氧化氫（H2O2）。
本論文接著探討兩個細胞凋亡的主要因子：粒線體膜電位（mitochondrial transmembrane potential, DYm）的下降及caspase 3/7的活化在sevoflurane處理後的影響，當細胞被處理sevoflurane後一小時就會出現粒線體膜電位與caspase 3/7的活化。再以彗星試驗（comet assay）和利用流式細胞儀（flow cytometry）去分析annexin V-FITV protein結合到細胞膜表面的量來證明sevoflurane誘導PMN細胞凋亡的作用。多型態核白血球（PMN）隨著暴露在不同濃度sevoflurane之下會增加細胞凋亡且會隨著濃度的增加而增加。總結，這些結果證實了sevoflurane處理人類多型態核白血球（human polymorphonuclear neutrophils, PMN）會造成氧化壓力（oxidative stress）及細胞傷害（cellular injury）。

Sevoflurane is an inhalation anesthetic used for general anesthesia. Several studies have demonstrated that reactive oxygen species (ROS) exist in cardioprotection when preconditioned with sevoflurane. Moreover, sevoflurane can also directly trigger the formation of peroxynitrite. Up to now, information pertinent to the effect of sevoflurane on cellular injuries in human polymorphonuclear neutrophils (PMN) is scanty. In this study, we demonstrated that sevoflurane significantly increases intracellular H2O2 and/or peroxide, superoxide, and nitric oxide (NO) in PMN within 1 h treatment. Intensification of intracellular glutathione (GSH) depletion in PMN has been demonstrated with the presence of sevoflurane. Inhibition of sevoflurane-mediated intracellular H2O2 and/or peroxide in PMN by catalase, mannitol, dexamethasone, N-acetylcysteine (NAC) and trolox, but not superoxide dismutase (SOD) pretreatment, was observed. Among them, catalase has the best effect scavenging intracellular H2O2 and/or peroxide, suggesting that H2O2 is the major ROS during sevoflurane treatment. Two apoptotic critical factors—lowering of the mitochondrial transmembrane potential and activation of caspase 3/7—were significantly increased after 1 h of sevoflurane treatment. Apoptosis of PMN were determined by comet assay and flow cytometric analysis of annexin V-FITV protein binding to the cell surface. Exposure of PMN to sevoflurane markedly increased apoptosis in a dose-dependent manner. In summary, these results are important for demonstrating the oxidative stress and cellular injury on sevoflurane-treated human PMN.

目 錄

中文摘要 -------------------------------------------------------------------------- ii
英文摘要 -------------------------------------------------------------------------- iv
目錄 -------------------------------------------------------------------------- vi
表目錄 -------------------------------------------------------------------------- vii
圖目錄 -------------------------------------------------------------------------- viii
符號說明 -------------------------------------------------------------------------- ix
第一章 緒論-------------------------------------------------------------------- 1
第二章 文獻探討-------------------------------------------------------------- 3
第一節 Sevoflurane的藥物特性-------------------------------------------- 3
第二節 就目前文獻上對於Sevoflurane與自由基相關的研究-------- 5
第三節 人類多型態核白血球與自由基的相關性 6
第四節 自由基------------------------------------------------------------------ 8
第五節 活性氧物質------------------------------------------------------------ 11
第六節 活性氧物質對人體的影響------------------------------------------ 13
第七節 人體內的抗氧物質-------------------------------------------------- 14
第八節 細胞凋亡(apoptosis)------------------------------------------------- 16
第九節 細胞凋亡(apoptosis)與自由基(Free radical)的相關性------- 19
第三章 研究方法-------------------------------------------------------------- 21
第一節 材料--------------------------------------------------------------------- 21
第二節 實驗方法--------------------------------------------------------------- 22
第四章 研究結果-------------------------------------------------------------- 32
第五章 討論與結論----------------------------------------------------------- 39
參考文獻 -------------------------------------------------------------------------- 48
圖表 -------------------------------------------------------------------------- 57
附錄 已發表之文獻-------------------------------------------------------- 69

參考文獻
Allaouchiche B, Debon R, Goudable J, Chassard D, Duflo F, 2001. Oxidative stress status during exposure to propofol, sevoflurane and desflurane. Anesth Analg .93,981-985.
Amer, J., Fibach, E., 2005. Chronic oxidative stress reduces the respiratory burst response of neutrophils from beta-thalassaemia patients. Br. J. Haematol. 129, 435-441.
Arriero MM, Munoz Alameda L, Lopez-Farre A, Escribano Burgos M, Carrasco C, Millas I, Celdran A, de la Pinta JC,2002. Sevoflurane reduces endothelium-dependent vasorelaxation: role of superoxide anion and endothelin. Can J Anaesth. 49(5),471-476.
Balaban, R.S., Nemoto, S., Finkel, T., 2005. Mitochondria, oxidants, and aging. Cell 120, 483-495.
Barry Halliwell, 1Matthew Whiteman.,2004. Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean? Br J Pharmacol, 142, 231–255.
Beaver, J.P., Waring, P. A., 1995. Decrease in intracellular glutathione concentration precedes the onset of apoptosis in murine thymocytes. Eur. J. Biol. 68, 47-54.
Bouwman, R.A., Musters, R.J., van Beek-Harmsen, B.J., de Lange, J.J., Boer, C., 2004. Reactive oxygen species precede protein kinase C-delta activation independent of adenosine triphosphate-sensitive mitochondrial channel opening in sevoflurane-induced cardioprotection. Anaesthesiol 100, 506-514.
Braga, P.C., dal Sasso, M., Bovio, C., Sgaragli, G.P., 2003. Effect of butylated hydroxyanisole and some of its derivatives on human neutrophil oxidative burst: chemiluminescence evaluation. Pharmacol 68, 9-16.
Brand, M.D., Murphy, M.P., 1987. Control of electron flux through the respiratory chain in mitochondria and cells. Biol. Rev. Camb. Philos. Soc., 62,141-193.
Burrows, D.L., Nicolaides, A., Stephens, G.C., Ferslew, K.E., 2004. The distribution of sevoflurane in a sevoflurane induced death. J Forensic Sci. 49, 1-4.
Chanock, S. J., Benna, J. E., Smith, R. M. and Babior,B. M. (1994) The respiratory burst oxidase. J. Biol.Chem., 269, 24519–24522.
Camilleri Broet S, Vanderwerff H, Caldwell E, et al.,1998. Distinct alterations in mitochondrial mass and function characterize different models of apoptosis. Exp Cell Res, 239 , 277-292.
Canakci, C.F., Cicek, Y., Canakci, V., 2005. Reactive oxygen species and human inflammatory periodontal diseases. Biochem (Mosc). 70, 619-628.
Chen, S.C., Kao, C.M., Huang, M.H., Shih, M.K., Chen, Y.L., Huang, S.P., Liu, T.Z., 2003. Assessment of genotoxicity of benzidine and its structural analogues to human lymphocytes using comet assay. Toxicol. Sci. 72, 283-288.
Chen, Z.H., Na, H.K., Hurh, Y.J., Surh, Y.J., 2005. 4-Hydroxyestradiol induces oxidative stress and apoptosis in human mammary epithelial cells: possible protection by NF-kappaB and ERK/MAPK. Toxicol. Appl. Pharmacol. 208, 46-56.
Derouet-Humbert, E., Roemer, K., Bureik, M., 2005. Adrenodoxin (Adx) and CYP11A1 (P450scc) induce apoptosis by the generation of reactive oxygen species in mitochondria. Biol. Chem. 386, 453-461.
Dworschak, M., Breukelmann, D., Hannon, J.D., 2004. Isoflurane applied during ischemia enhances intracellular calcium accumulation in ventricular myocytes in part by reactive oxygen species. Acta. Anaesthesiol. Scand. 48, 716-721.
Green DR, Reed JC., 1998 Mitochondria and apoptosis. Science, 281, 1309-1312.
Hadjigogos, K., 2003. The role of free radicals in the pathogenesis of rheumatoid arthritis. Panminerva Med. 45, 7-13.
Heiskanen, K.M., Naarala, J., Savolainen, K.M., 1995. The effects of N-(5-vinyl-1,3-thiazolidin-2-ylidene)phenylamine (5-VTPA) on the changes in free intracellular calcium and the production of reactive oxygen metabolites in human leukocytes. Toxicol 100, 195-202.
Higuchi, Y., 2004. Glutathione depletion-induced chromosomal DNA fragmentation associated with apoptosis and necrosis. J. Cell. Mol. Med. 8, 455-464.
Hsieh, T.J., Liu, T.Z., Chern, C.L., Tsao, D.A., Lu, F.J., Syu, Y.H., Hsieh, P.Y., Hu, H.S., Chang, T.T., Chen, C.H., 2005. Liriodenine inhibits the proliferation of human hepatoma cell lines by blocking cell cycle progression and nitric oxide-mediated activation of p53 expression. Food Chem. Toxicol. 43, 1117-1126.
Hsieh, T.J., Liu, T.Z., Chia, Y.C., Chern, C.L., Lu, F.J., Chuang, M.C., Mau, S.Y., Chen, S.H., Syu, Y.H., Chen, C.H., 2004. Protective effect of methyl gallate from Toona sinensis(Meliaceae) against hydrogen peroxide-induced oxidative stress and DNA damage in MDCK cells. Food Chem. Toxicol. 42, 843-850.
James F. Curtin, Maryanne Donovan , Thomas G. Cotter.,2002. Regulation and measurement of oxidative stress in apoptosis.J Immunol Methods, 265, 49-72.
Kanna, T., Akata, T., Izumi, K., Nakashima, M., Yonemitsu, Y., Hashizume, M., Takahashi, S., 2002. Sevoflurane and bradykinin-induced calcium mobilization in pulmonary arterial valvular endothelial cells in situ: sevoflurane stimulates plasmalemmal calcium influx into endothelial cells. J. Cardiovasc. Pharmacol. 40, 714-724.
Kikugawa, K., Oikawa, N., Miyazawa, A., Shindo, K., Kato, T., 2005. Interaction of nitric oxide with glutathione or cysteine generates reactive oxygen species causing DNA single strand breaks. Biol. Pharm. Bull. 28, 998-1003.
Kito, M., Akao, Y., Ohishi, N., Yagi, K., Nozawa, Y., 2002. Arsenic trioxide-induced apoptosis and its enhancement by buthionine sulfoximine in hepatocellular carcinoma cell lines. Biochem. Biophys. Res. Commun. 291, 861-867.
Kopprasch, S., Pietzsch, J., Graessler, J., 2004. The protective effects of HDL and its constituents against neutrophil respiratory burst activation by hypochlorite-oxidized LDL. Mol. Cell. Biochem. 258, 121-127.
Kovacic, P., Thurn, L.A., 2005. Cardiovascular toxicity from the perspective of oxidative stress, electron transfer, and prevention by antioxidants. Curr. Vasc. Pharmacol. 3, 107-117.
Kwon, K.B., Kim, E.K., Lim, J.G., Shin, B.C., Han, S.C., Song, B.K., Kim, K.S., Seo, E.A., Ryu, D.G., 2005. Protective effect of Coptidis Rhizoma on S-nitroso-N-acetylpenicillamine (SNAP)-induced apoptosis and necrosis in pancreatic RINm5F cells. Life Sci. 76, 917-929.
Larochette, N., Decaudin, D., Jacotot, E., Brenner, C., Marzo, I., Susin, S.A., Zamzami, N., Xie, Z., Reed, J., Kroemer, G., 1999. Arsenite induces apoptosis via a direct effect on mitochondrial permeability transition pore. Exp. Cell Res. 249, 413-421.
Lavi, R., Shainberg, A., Friedmann, H., Shneyvays, V., Rickover, O., Eichler, M., Kaplan, D., Lubart, R., 2003. Low energy visible light induces reactive oxygen species generation and stimulates an increase of intracellular calcium concentration in cardiac cells. J. Biol. Chem. 278, 40917-40922.
Lee, C.S., Kim, Y.J., Ko, H.H., Han, E.S., 2005. Synergistic effects of hydrogen peroxide and ethanol on cell viability loss in PC12 cells by increase in mitochondrial permeability transition. Biochem. Pharmacol. 70, 317-325.
Lin, Y., Berg, A.H., Iyengar, P., Lam, T.K., Giacca, A., Combs, T.P., Rajala, M.W., Du, X., Rollman, B., Li, W., Hawkins, M., Barzilai, N., Rhodes, C.J., Fantus, I.G., Brownlee, M., Scherer, P.E., 2005. The hyperglycemia-induced inflammatory response in adipocytes: the role of reactive oxygen species. J. Biol. Chem. 280, 4617-4626.
Machida, K., Tanaka, T., 1999. Farnesol-induced generation of reactive oxygen species dependent on mitochondrial transmembrane potential hyperpolarization mediated by F(0)F(1)-ATPase in yeast. FEBS Lett. 462, 108-112.
Malviya, S., Voepel-Lewis, T., Tait, A.,Reynolds, P.I., Gujar, S.K., Gebarski, S.S., Petter Eldevik, O., 2004. Pentobarbital vs choral hydrate for sedation of children undergoing MRI: efficacy and recovery characteristics. Paediatr. Anaesth.14, 589-595.
Nathan, C. (1992) Nitric oxide as a secretory product of mammalian cells. FASEB J., 6, 3051–3064.
Niemczyk, E., Majczak, A., Hallmann, A., Kedzior, J., Wozniak, M., Wakabayashi, T., 2004. A possible involvement of plasma membrane NAD(P)H oxidase in the switch mechanism of the cell death mode from apoptosis to necrosis in menadione-induced cell injury. Acta. Biochim. Pol. 51, 1015-1022.
Nussler, A. K. and Billiar, T. R. (1993) Inflammation,immunoregulation, and inducible nitric oxidesynthase. J. Leukoc. Biol., 54, 171–178.
O''Neill, A.J., O''Neill, S., Hegarty, N.J., Coffey, R.N., Gibbons, N., Brady, H., Fitzpatrick, J.M., Watson, R.W., 2000. Glutathione depletion-induced neutrophil apoptosis is caspase 3 dependent. Shock. 14, 605-609.
van Oostrom, A.J., van Wijk, J., Cabezas, M.C., 2004. Lipaemia, inflammation and atherosclerosis: novel opportunities in the understanding and treatment of atherosclerosis. Drugs 64, 19-41.
Peter ME, Heufelder AE, Hengartner MO., 1997. Advances in apoptosis research. Proc Natl Acad Sci USA, 94, 12736-12737.
Perl., A, Nagy, G., Gergely, P., Puskas, F., Qian, Y., Banki, K., 2004. Apoptosis and mitochondrial dysfunction in lymphocytes of patients with systemic lupus erythematosus. Methods Mol. Med. 102, 87-114.
Porter, A,G., Jänicke, R.U., 1999. Emerging roles of caspase-3 in apoptosis. Cell Death Differ. 6, 99-104.
Rego, A.C., Oliveira, C.R., 2003. Mitochondrial dysfunction and reactive oxygen species in excitotoxicity and apoptosis: implications for the pathogenesis of neurodegenerative diseases. Neurochem. Res. 28, 1563-1574.
Reinhardt-Poulin, P., McLean, J.R., Deslauriers, Y., Gorman, W., Cabat, S., Rouabhia, M., 2000. The use of silver-stained "comets" to visualize DNA damage and repair in normal and Xeroderma pigmentosum fibroblasts after exposure to simulated solar radiation. Photochem. Photobiol. 71, 422-425.
Riess, M.L., Eells, J.T., Kevin, L.G., Camara, A.K., Henry, M.M., Stowe, D.F., 2004. Attenuation of mitochondrial respiration by sevoflurane in isolated cardiac mitochondria is mediated in part by reactive oxygen species. Anesthesiol 100, 498-505.
Riess, M.L., Kevin, L.G., McCormick, J., Jiang, M.T., Rhodes, S.S., Stowe, D.F., 2005. Anesthetic preconditioning: the role of free radicals in sevoflurane-induced attenuation of mitochondrial electron transport in Guinea pig isolated hearts. Anesth. Analg. 100, 46-53.
de Ruijter, W., Musters, R.J., Boer, C., Stienen, G.J., Simonides, W.S., de Lange, J.J., 2003. The cardioprotective effect of sevoflurane depends on protein kinase C activation, opening of mitochondrial K(+)(ATP) channels, and the production of reactive oxygen species. Anesth. Analg. 97, 1370-1376.
Sharma, P., Raghavan, S.A., Saini, R., Dikshit, M., 2004. Ascorbate-mediated enhancement of reactive oxygen species generation from polymorphonuclear leukocytes: modulatory effect of nitric oxide. J. Leukoc. Biol. 75, 1070-1078.
Spector, A., 1995. Oxidative stress-induced cataract: Mechanism of action. FASEB J. 9, 1173-1182.
Stadtman, E.R., Van Remmen, H., Richardson, A., Wehr, N.B., Levine, R.L., 2005. Methionine oxidation and aging. Biochim. Biophys. Acta. 1703, 135-140.
Szocs, K., 2004. Endothelial dysfunction and reactive oxygen species production in ischemia/reperfusion and nitrate tolerance. Gen. Physiol. Biophys.; 23: 265-295.
Tay, V.K., Wang, A.S., Leow, K.Y., Ong, M.M., Wong, K.P., Boelsterli, U.A., 2005. Mitochondrial permeability transition as a source of superoxide anion induced by the nitroaromatic drug nimesulide in vitro. Free Radic Biol Med. 39, 949-959.
Thornberry NA, Lazebnik Y.,1998. Caspases: enemies within. Science, 281, 1312-1316.
Tripathy, D., Mohanty, P., Dhindsa, S., Syed, T., Ghanim, H., Aljada, A., Dandona, P., 2003. Elevation of free fatty acids induces inflammation and impairs vascular reactivity in healthy subjects. Diabetes 52, 2882-2887.
Valko, M., Izakovic, M., Mazur, M., Rhodes, C.J., Telser, J., 2004. Role of oxygen radicals in DNA damage and cancer incidence. Mol. Cell Biochem. 266, 37-56.
Walrand, S., Farges, M., Dehaese, O., Cardinault, N., Minet-Quinard, R., Grolier, P., Bouteloup-Demange, C., Ribalta, J., Winklhofer-Roob, B.M., Rock, E., Vasson, M.P., 2005. In vivo and in vitro evidences that carotenoids could modulate the neutrophil respiratory burst during dietary manipulation. Eur. J. Nutr. 44,114-120.
Wink, D. A. and Mitchell, J. B. (1998) Chemical biology of nitric oxide:

Insights into regulatory,cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radic. Biol. Med., 25, 434–45
Yoshida K-I, Okabe E, 1992. Selective impairment of endothelium-dependent relaxation by sevoflurane: Oxygen free radicals participation. Anesthesiology. 76,440-447.
Yu, B.P., 1994. Cellular defenses against damage from reactive oxygen species. Physiol. Rev. 74, 139-162.