臺灣博碩士論文加值系統

越來越多研究文獻顯示,當陽光中的紫外線照射我們的皮膚細胞會引發一些細胞傷害。一般普遍認同會加速皮膚老化，及造成皮膚一些相關疾病，主要因素是活性氧(ROS; Reactive oxygen species)所造成的。本研究是利用人類皮膚纖維母細胞(Human Foreskin Fibroblast)為實驗對象，利用純化的propyl gallate (PG)藉以研究它是否具有保護皮膚細胞抗紫外線傷害的作用。由實驗結果了解到紫外線(UVA; 50KJ/cm2)對細胞帶來的傷害像是讓細胞存活度降低外，還包括細胞內氧化壓力上升，而抗氧化系統例如胞內GSH之耗損。當我們加入不同濃度的PG於細胞培養液中後我們發現PG可以: (1)提升紫外線照射細胞後的細胞存活度; (2)有效地降低細胞內ROS生成(PG在最低濃度10ug/ml就有效果); (3)保護細胞內GSH缺損情況緩解; (4)除此之外，亦可以改善UVA造成的lipid oxidation及降低UVA造成細胞之程式死亡。綜合言之，目前的實驗結果初步顯示，PG確實可以促進皮膚纖維母細胞增強其防護紫外線損害之功效。

There is increasing evidence to substantiate that reactive oxygen species (ROS) are intimately involved in the oxidative damage of human skin cells following the exposure of these cells to UVA irradiation. Thus, it is desirable to search for chemopreventive agents that can counteract ROS-mediated injury to the skin cells. The specific aim of this study is thus to evaluate whether or not propyl gallate (PG), a dietary supplement, could offer protection against UVA-evoked oxidative damage of human fibroblasts. Using human foreskin fibroblast cells (HFF3) as the experimental cell model, we first demonstrated that when these cells were exposed to various fluences of UVA-irradiation, a concentration- dependent impediment of cell growth resulted. The demise of UVA- evoked cell death was shown to be apoptotic in nature, as reflected by a fluence-dependent increases in TUNEL-positive cells. The cascade of events of UVA-evoked apoptotic cell death were shown to be involved in the initial overproduction of ROS, followed by the occurrence of intracellular glutathione (iGSH) depletion and lipid peroxidation. A nearly 80% of intracellular ROS generated when HFF3 cell were exposed to a fluence of 50KJ/cm2 UVA irradiation could be removed by PG (40ug/ml). Similarly, iGSH depletion and lipid peroxidation caused by UVA-
irradiation could also be protected effectively by PG. Collectively , we have presented experimental evidence here that PG could offer protective capability against UVA-induced oxidation damage via scavenging intracellular ROS, inhibiting GSH depletion, lipid peroxidation,mitochondrial depolarization and apoptotic cell death of HFF3 cells.
Thus, PG can be used as a chemopreventive agent against UVA-evoked oxidative damages, and therefore can be considered as a supplement in an anti-aging cosmetic product.The possibility warrants further investigation.

指導教授推薦書……………………………...…………………………
口試委員審定書…………………...……………………………………
授權書………………………………...…………………………………iv
誌謝…………………………………...…………………………………v
縮寫表…………………………………...…..…………………………vii
中文摘要…………………………………...…………………………..viii
英文摘要…………………………………………………………….....ix
目錄………………..…………………………………………………… xi
第一章 簡介…………………………………………………………... 1
1.1 紫外線簡介(Ultraviolet introduce).…............. 1
1.2 自由基產生與自由基誘導皮膚傷害(ROS production and ROS
induced skin damage)…………………………………………....6
1.2.1紫外線與自由基(UV and ROS).......................6
1.2.2膠原纖維的變性(collagen denature)...……...……….7
1.2.3彈性纖維的變性(elastin denature)…..…......………8
1.2.4細胞膜脂質過氧化(lipid peroxidation)….……….....8
1.2.5 DNA斷裂(DNA broken)…………..…..…...……….....9
1.2.6酵素及非酵素抗氧化系統破壞……………………………………10
1.2.6.1非酵素性抗氧化系統……………………………………………10
1.2.6.2酵素性抗氧化系統..……………………………………………11
1.2.7紫外線對於皮膚抗氧化系統之影響………………………………13
1.3 沒食子酸丙基簡介(propyl gallate introduce)……………14
1.4 研究目的(Specific Aims) ………………………………………16
第二章 材料與方法……………….....…………………………………18
2.1 實驗材料………………………………………………………………18
2.1.1 細胞株與細胞培養基………………………………………………18
2.1.2 其它藥品、緩衝液與試劑…………………………………………18
2.1.3 細胞計數………….……...………………………………………18
2.1.4 ROS偵測染劑………………………………………………………19
2.1.5 脂質過氧化測定之染劑……………………………………………19
2.1.6 GSH含量測定之染劑………………………………………………19
2.1.7 粒線體膜電位測定之染劑…………………………………………19
2.1.8 細胞週期測定染劑…………………………………………………19
2.1.9 測定細胞凋亡之kit………………………………………………19
2.1.10 西方墨點法所須抗體……………………………………………19
2.2 實驗方法………………………………………………………………20
2.2.1 Cell culture……………………………………………………20
2.2.2 Cell count………………………………………………………20
2.2.3 Pretreat propyl gallate……………………………………20
2.2.4 ROS test……………………………………………………………20
2.2.5 Cell cycle test ………………………………………………21
2.2.6 GSH test…….……………………………………………………21
2.2.7 Mitochondria potential test………….…………………21
2.2.8 lipid oxidation test…………………………………………22
2.2.9 TUNEL assay………………………………………………………22
2.2.10 Western Blot……………………………………………………23
第三章 結果…………………………………………………………………25
3.1 UV照射對纖維母細胞影響……………………………………………25
3.1.1 UV對細胞存活率的影響……………………………………………25
3.1.2 UV照射與細胞產生自由基的表現之關係…………………………25
3.1.3 UV照射與胞內GSH耗損之關係……………………………………26
3.1.4 UV照射產生細胞程式凋亡之偵測…………………………………26
3.2 propyl gallate 對細胞存活度影響………………………………26
3.3 PG 對於UV引起細胞傷害的影響…………………………………….26
3.3.1存活度的影響…………………………………………………………26
3.3.2 UVA照射與細胞內自由基之關係……………………………………27
3.3.3 UVA照射與細胞內GSH耗損之關係…………………………………27
3.3.4 UVA照射與細胞內lipid peroxidation之關係…………………27
3.3.5細胞內粒線體之膜電位之變化………………………………………28
3.3.6細胞凋亡之偵測………………………………………………………28
第四章 討論……………………………………………………………………29
4.1 紫外線對細胞的影響.…………………………………………………29
4.2 Propyl gallate 對細胞的影響……………………………………30
4.3 Propyl gallate與UVA照射對纖維母細胞的影響…………………30
第五章 結論………...……………………..………………………………34
第六章 參考文獻….……………………..…………………………………35
第七章 圖表….……………………..………………………………………42

Pahl HL, Baeuerle PA. Oxygen and the control of gene expression. .Bioessays. 16:497–502, 1994.

Ferguson HA, Marietta PM, Van Den Berg CL. UV-induced apoptosis is mediated independent of caspase-9 in MCF-7 cells: a model for cytochrome c resistance. THE JOURNAL OF BIOLOGICAL CHEMISTRY . 278: 45793–45800, 2003.

Bhawan J, Olsen E, Lufrano L, Thorne EG, Schwab B and Gilchrest BA. Histologic evaluation of the long term effects of tretinoin on photodamaged skin. J. Dermatol. Sci. 11: 177-182, 1996.

Hiroyuki Yasui, Hiromu Sakurai. Essential role of singlet oxygen species in cytochrome P450-dependent substrate oxygenation by rat liver microsomes. Biochemicaland Biophysical Research Communication. ,269(2):131-136, 2000.

Yasui H, Fujii S, Yoshimura T, Sakurai H. Spinnokinetic analyses of blood disposition and biliary excretion of nitric oxide (NO)-Fe(II)-N-(dithiocarboxy)sarcosine complex in rats: BCM-ESR and BEM-ESR studies. Free Radic Res. 38(10):1061-72, 2004.

Mittal A, Elmets CA, Katiyar SK. CD11b+ cells are the major source of oxidative stress in UV radiation-irradiated skin: possible role in photoaging and photocarcinogenesis. Photochem Photobiol. 77(3):259-264, 2003.

Fisher GJ. The pathophysiology of photoaging of the skin. Cutis, 75(2):5-8, 2005.

Sehrawat A, Sharma S, Sultana S. Preventive effect of tannic acid on 2-acetylaminofluorene induced antioxidant level, tumor promotion and hepatotoxicity: a chemopreventive study. Redox Rep. 11, 85–95, 2006.

Chen SC, Chung KT. Mutagenicity and antimutagenicity studies of tannic acid and its related compounds. Food Chem. Toxicol. 38: 1–5, 2000.

Szaefer H, Kaczmarek J, Rybczynska M, Baer-Dubowska W. The effect of plant phenols on the expression and activity of phorbol ester-induced PKC in mouse epidermis. Toxicology .230: 1–10, 2007.

Buttke TM, Sandstrom PA . Oxidative stress as a mediator of apoptosis. Immunol.Today 15: 7-10,1994.

Cai J, Yang J, Jones DP . Mitochondrial control of apoptosis: the role of cytochrome C. Biochim Biophys Acta 1366:139-149,1998.

Chan W-H, Yu J-S, Yang S-D . Heat shock stress induces cleavage and activation of PAK2 in apoptotic cells. J Protein Chem 17:485-494, 1998.

Chan W-H, Yu J-S, Yang S-D . PAK2 is cleaved and activation during hyperosmotic shock-induced apoptosis via a caspase-
dependent mechanism. Evidence for the involvement of oxidative stress. J Cell Physiol 178: 397-408, 1999.

Chan and Yu . Inhibition of UV irradiation-induced oxidative stresses and apoptotic biochemical changes in human epidermal carcinoma A431 cells by genistein. J Cell Biochem 78:73-84, 2000.

Ellis RE, Yuan J-Y, Horvitz HR . Mechanisms and functions of cell death. Ann Rev Cell Biol 7: 663-698,1991.

Green DR, Reed JC . Mitochondria and apoptosis. Science 281: 309-1312, 1998.

Jacobson, M. D., Weil, M., and Raff, M. C. Programmed cell death in animal development. Cell 88:347-354, 1997.

Lenaz G, Bovina C, Formiggini G, Castelli GP . Mitochondria, oxidative stress and antioxidant defences. Acta Biochim Pol 46:1-21, 1999

Thiele J, Elsner P. Oxidants and Antioxidants in Cutaneous Biology. Curr Probl Dermatol. 29:83-94, 2001.

Christine D, Isabelle K. Induction of thioredoxin by ultraviolet-A radiation prevents oxidative-mediated cell death in human skin fibroblast. Free Radical Biology&Medicine. 31:585-598, 2001.

Cecilia A.B., Petra K.L., Katarina M.K. UVA/B induced apoptosis in human melanocytes involves translocation of Cathepsins and Bcl-2 family members. Jounal of Investigative Dermatology. 52:937-945, 2002.

Ching H.C., Tsan Z.L., Chin H.C., Chung H.W., Chi H.C., Fung J.L. The efficacy of protective effects of tannic acid, gallic acid, ellagic acid, and propyl gallate against hydrogen peroxide-induced oxidative stress and DNA damages in IMR-90 cells. Mol.Nutr.Food.Res. 51:962-968, 2007.

Eung R.L., Jung H.K., Yong J.K., Ssang G.C. The anti-apoptotic and anti-oxidant effect of Eriodictyol on UV-induced apoptosis in Keratinocytes. Biol. Pharm. Bull. 30:32-37, 2007.

Alena Svobodova, Daniela Walterova, Jitka Vostalova. Ultraviolet light induced alteration to the skin. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 150(1):25-38, 2006.

Matthias S.,Hue-Tran Hornig-Do, Sarah Schomberg, Gernot Herrmann, Rudolf J. Mitochondrial electron transport chain activity is not involved in ultraviolet A (UVA)-induced cell death. Free Radical Biology & Medicine. 42:499-509, 2007.



Hirokazu W., Tadamichi S.,Jun N. Ultraviolet A-induced production of Matrix Metalloproteinase-1 is mediated by macrophage migration inhibitory factor (MIF) in human dermal fibroblast. The Journal Of Biological Chemistry. 279(3): 1676-1683, 2004.

Antonio V., Anpuchchelvi R., A. Bjorn Carle, Irene E. Kochevar. 7-Dehydrocholesterol enhances ultraviolet A-induced oxidative stress in keratinocytes: Roles of NADPH oxidase, mitochondria, and lipid rafts. Free Radical Biology & Medicine 41:1704-1718,2006.

Laure Rittie’, Gary J. Fisher. UV-light-induced signal cascades and skin aging. Ageing Research Reviews. 1:705-720, 2002.

Herrmann G., Wlaschek M., Lange TS., Prenzel K.,Goerz G., Scharffetter-Kochanek K. UVA irradiation stimulates the synthesis of various matrix-metalloproteinases (MMPs) in cultured human fibroblast. Exp Dermatol. 2(2):92-97, 1993.

Scharffetter K, Wlaschek M, Hogg A, Bolsen K, Schothorst A, Goerz G, Krieg T. UVA irradiation induces collagenase in human dermal fibroblasts in vitroand in vivo. Arch Dermatol Res. 283(8): 506-511, 1991.

Naru E., Suzuki T., Moriyama M., Inomata K., Hayashi A., Arakane K., Kaji K. Functional changes induced by chronic UVA irradiation to cultured human dermal fibroblasts. Br J Dermatol. 2: 6-12, 2005.

Toblas P., Rex M. Tyrrell. Involvement of lipid peroxidation and organic peroxides in UVA-induced matrix metalloproteinase-1 expression. Free Radical Biology & Medicine. 36: 1566-1575, 2004.

Yu-Ying H., Jian-li H., Colin F. C. Delayed and sustained activation of extracellular signal-regulated kinase in human keratinocytes by UVA. The Journal Of Biological Chemistry. 279:53867-53874, 2004.

Ming K.S., Ching H.C., Tsan Z.L. Exacerbation of UVA-induced mitochondrial dysfunction of human SK-Hep-1 cells by Ferrous Ions. J Biomed Lab Sci .13:24-28, 2001.

Bor-Show T., Yun-Jung C., Hsin-Chieh L., Chu-Bin L. Tuning up or down the UV-induced apoptosis in chinses hamster ovary cells with cell cycle inhibitors. Photochemistry and Photobiology. 75(6): 662-667, 2002.

Sehrawat, A., Sharma, S., Sultana, S., Preventive effect of tannic acid on 2-acetylaminofluorene induced antioxidant level, tumor promotion and hepatotoxicity: A chemopreventive study. Redox Rep. 11: 85–95, 2006.

Wu, L. T., Chu, C. C., Chung, J. G., Chen, C. H. et al., Effects of tannic acid and its related compounds on food mutagens or hydrogen peroxide-induced DNA strands breaks in human lymphocytes. Mutat. Res. 556: 75–82, 2004.

Chen, S. C., Chung, K. T., Mutagenicity and antimutagenicity
of tannic acid and its related compounds. Food Chem. Toxicol.38: 1–5, 2000.

Kuo, M. L., Lee, K. C., Lin, J. K., Genotoxicities of nitropyrenes and their Modulation by apigenin, tannic acid, ellagic acid and indole-carbinol in Salmonella and CHO systems. Mutat. Res. 270: 87 –95, 2000.

Szaefer, H., Kaczmarek, J., Rybczynska, M., Baer-Dubowska,
W., The effect of plant phenols on the expression and activity of phorbol ester-induced PKC in mouse epidermis. Toxicology 230:1–10, 2007.

Loarca-Pina, G., Kuzmicky, P. A., Gonzalez de Mejia, E., Kado, N. Y. et al., Antimutagenicity of ellagic acid against aflatoxin B1 in the Salmonella microsuspension assay. Mutat.Res. 360: 15–21, 1996.

Loarca-Pina, G., Kuzmicky, P. A., de Mejia, E. G., Kado, N.
Y., Inhibitory effects of ellagic acid on the direct-acting mutagenicity of aflatoxin B1 in the Salmonella microsuspension assay. Mutat. Res. 398:183–187, 1998.

Wang, Z. Y., Cheng, S. J., Zhou, Z. C., Athar, M. et al., Antimutagenicity activity of green tea polyphenols. Mutat. Res.223:273 –285, 1989.

Falsig, J., Christiansen, S. H., Feuerhahn, S., Burkle, A. et al.,Poly(ADP-ribose) glycohydrolase as a target for neuroprotective intervention: Assessment of currently available pharmacological tools. Eur. J. Pharmacol. 497: 7–16, 2004.

Khan, N. S., Ahmad, A., Hadi, S. M., Anti-oxidant, prooxidant properties of tannic acid and its binding to DNA. Chem. Biol. Interact. 125: 177 –189, 2000.

Labieniec, M., Gabryelak, T., Falcioni, G., Antioxidant and
prooxidant effects of tannins in digestive cells of the freshwater mussel Unio tumidus. Mutat. Res. 539: 19–28, 2003.

Tsai, C. H., Chern, C. L., Liu, T. Z., Antioxidant action of glutathione: Its reaction with superoxide anion and hydroxyl radicals. J. Biomed. Lab. Sci. 12: 107 –111, 2000.

Munday, R., Winterbourn, C. C., Reduced glutathione in combination with superoxide dismutase as an important biological antioxidant defense mechanism. Biochem. Pharmacol.38: 4349 –4352, 1989.

Winterbourn, C. C., Metodiewa, D., The reaction of superoxide with reduced glutathione. Arch. Biochem. Biophys. 314: 284–290, 1994.



Yamauchi, N., Watenabe, N., Kuriyama, H., Suppressive
effects of intracellular glutathione on hydroxyl radical production induced by tumor necrosis factor. Int. J. Cancer. 46: 884–886, 1990.

McNeil, C. J., Banford, J. C., Brown, D. H., A relationship
between thiols and the superoxide ion. FEBS Lett. 133:175–179,1981.

Karihtala, P., Soini, Y., Reactive oxygen species and antioxidant mechanisms in human tissues and their relation to malignancies.APMIS . 115: 81–103, 2007.

Bahar, G., Feinmesser, R., Shpitzer, T., Popovtzer, A., Nagler,R. M., Salivary analysis in oral cancer patients: DNA and protein oxidation, reactive nitrogen species, and antioxidant profile.Cancer . 109: 54–59, 2007.