跳到主要內容

臺灣博碩士論文加值系統

(3.236.50.201) 您好!臺灣時間:2021/08/06 08:59
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:詹桂榕
研究生(外文):Kuei-Jung Chan
論文名稱:白頭翁素抑制人類正常黑色素細胞內酪氨酸酵素活性與酪氨酸相關酵素表現
論文名稱(外文):Anemonin inhibits the tyrosinase activity and expression oftyrosinase-related proteins in human epidermal melanocytes
指導教授:李美賢李美賢引用關係
指導教授(外文):Mei-Hsien Lee
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:73
中文關鍵詞:黑色素細胞黑色素酪氨酸酵素白頭翁素酪氨酸相關酵素美白
外文關鍵詞:melanocytetyrosinasetyrosinase-related proteinsanemoninmelanindepigmentation
相關次數:
  • 被引用被引用:1
  • 點閱點閱:305
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:1
在黑色素生成步驟裡酪氨酸相關酵素共同參與其反應過程,包含酪氨酸酵素、酪氨酸相關酵素1和酪氨酸相關酵素2。酪氨酸酵素是個含銅的單氧酶,在生成黑色素中進行羥基化與氧化反應此為合成黑色素的速率決定步驟。
本實驗的20種純化合物由天然物分離出來,包含植物、黴菌、海洋細菌與海藻,拿來篩選對黑色素細胞內酪氨酸酵素的活性與抑制黑色素生成的效果。在實驗過程中,一開始化合物在50 µM的濃度時處理正常人類黑色素細胞24小時,得到結果為10種化合物的細胞存活率>80%。此10種化合物接下來也以50 µM進行對酪氨酸酵素活性的探討。在此步實驗結果顯示,白頭翁素(Anemonin)對酪氨酸酵素的抑制活性能大於50%。
由上述的篩選過程中發現白頭翁素(Anemonin)由厚葉鐵線蓮(Clematis crassifolia Bentham)萃取而來,具有抑制黑色素細胞內酪氨酸酵素的能力其IC50為43.5 µM,當細胞加藥處理48小時,酪氨酸酵素抑制率可高達60%以上。在抑制黑色素生成方面,加藥24小時後對黑色素的生成並無明顯的影響,但持續加藥48小時後黑色素生成可將低約70%左右。
因此在確定黑色素被抑制後進一步的討論白頭翁素對黑色素細胞體內機制的探討,在西方點墨法的結果酪氨酸酵素與相關酵素2的蛋白表現在24小時後都被明顯的抑制,但酪氨酸相關酵素1的蛋白表現在48小時後才有明顯的抑制。以細胞免疫染色法加以觀測細胞內此3種酵素,酪氨酸酵素與酪氨酸相關酵素2在細胞體內的表現是降低的。最後利用即時定量聚合酵素連鎖反應觀察3種酵素的基因表現,白頭翁素確實能減少酪氨酸及其相關蛋白的基因表現。
本實驗結果討論,白頭翁素在影響黑色素的生成原因可能是經由抑制酪氨酸及其相關酵素的基因表現,因此能調控此3種酵素的蛋白表現。白頭翁素對黑色素細胞在不產生細胞毒性之下能夠抑制黑色素的生成,但完整的作用機制尚未明確,希望在未來可以實際運用在化妝品美白的用途上,做為新一代皮膚美白劑。
Melanin synthesis is a highly cooperative process carried out by tyrosinase family proteins, including tyrosinase, tyrosinase-related protein 1 (TRP1) and tyrosinase-related protein 2 (TRP2). Tyrosinase is a copper-containing monooxygenase. Tyrosinase catalysis reaction including hydroxylation and oxidation steps which are the rate-limiting steps in melanin production.
In our study, 20 compounds isolated from natural products, including plants, fungus, ocean bacteria and seaweed, were evaluated their ability to inhibit cellular tyrosinase activity and reduce melanin synthesis in human epidermal melanocyte (HEMn).
Among the compounds, cell viabilities of 10 compounds were>80%, when cells were treated with 50 µM of different compounds for 24 hours. They were further evaluated the tyrosinase activity. Among these compounds, Anemonin isolated from Clematis crassifolia Bentham showed the most efficacious in inhibition of tyrosinase activity. When treated with Anemonin for 48 hours, the inhibition of tyrosinase activity was above 60%. Melanin synthesis was reduced to 70% when sustained administration for 48 hours. But it showed no variation in melanin synthesis when administration for 24 hours.
The cellular mechanism in melanocyte was further investigated. The protein expression of tyrosinase and TRP2 were significantly Inhibited in 24 hours but the protein expression of TRP1 were inhibited until 48 hours later. Decreased of these protein expression were further confirmed by immunocytochemistry. Anemonin suppressed the expression of tyrosinase and it’s related proteins as demonstrated by quantitative real-time PCR (qRT-PCR).
In conclusion, Anemonin may down-regulated gene encoding of tyrosinase and it’s related proteins (TRP2 and TRP2) and resulted in reducing melanin synthesis. Anemonin can inhibit melanin synthesis without cytotoxicity in human melanocyte although cellular mechanism was still unclear. Our results suggest that Anemonin can be used as whitening agent for therapeutic intervention of hyperpigmentation on cosmetics.
縮寫表...................................................i
中文摘要................................................ii
英文摘要................................................iv
第一章 緒論..............................................1

1.1 黑色細胞與相關介紹...................................2
1.1.1 胚胎學.............................................2
1.1.2 黑色素細胞.........................................2
1.1.3 黑色素生成.........................................2
1.1.4 酪氨酸酵素.........................................3
1.1.5 抑制黑色素生成的方法...............................4
1.1.6 熊果素介紹.........................................5
1.2 化合物介紹...........................................6
1.3 Anemonin (Cpd8, 白頭翁素)介紹........................9
1.4 研究動機............................................10

第二章 材料與實驗方法...................................11
2.1.1一般藥品與試劑.....................................12
2.1.2 細胞株............................................13
2.1.3 細胞培養液........................................13
2.1.4 一級抗體..........................................13
2.1.5 二級抗體..........................................13
2.1.6 機器設備..........................................14
2.1.7 分析軟體..........................................14
2.2 人類黑色素細胞培養..................................15
2.3 細胞存活率試驗......................................15
2.4 蛋白定量............................................16
2.5 細胞內酪氨酸酵素活性................................16
2.6 黑色素細胞中黑色素生成測量..........................17
2.7 酪氨酸酵素氧化活性染色法............................17
2.8西方墨點法...........................................18
2.9 免疫螢光染色........................................18
2.10 RNA萃取與聚合酵素連鎖反應.........................19
2.10.1 Total RNA萃取....................................20
2.10.2 RNA定量..........................................20
2.10.3 cDNA製程.........................................21
2.11 即時定量聚合酵素連鎖反應...........................22
2.12 藥物製備...........................................23
2.13 統計方法...........................................23

第三章 實驗結果.........................................24
3.1 細胞存活率試驗......................................25
3.2 細胞酪氨酸酵素活性試驗..............................25
3.3 Anemonin (Cpd8)不同濃度對細胞內酪氨酸酵素的活性.....26
3.4 Anemonin (Cpd8)不同時間點對細胞內酪氨酸酵素的活性...26
3.5 Anemonin (Cpd8)不同濃度對細胞中黑色素生成測量.......26
3.6 Anemonin (Cpd8)不同時間點對細胞中黑色素生成測.......27
3.7 以活性染色法觀測Anemonin (Cpd8)不同濃度對酪氨酸酵素氧化
活性作用............................................27
3.8 Anemonin (Cpd8)影響黑色素細胞內酪氨酸相關蛋白表現...28
3.9 利用免疫細胞染色法偵測Anemonin (Cpd8)對黑色素細內酪氨酸
酵素相關蛋白表現....................................29
3.10Anemonin (Cpd8)影響黑色素細胞內酪氨酸相關蛋白的mRNA表
現..................................................29

第四章 討論.............................................31
第五章 結論.............................................37
參考文獻................................................40
1.蘇信義. 基礎皮膚科學. 1983.
2.Del Marmol V., Beermann F. Tyrosinase and related
proteins in mammalian pigmentation. FEBS Lett,1996.381
(3): p.165-168.
3.Seo S. Y., Sharma V. K., Sharma N. Mushroom tyrosinase:
recent prospects. J Agric Food Chem,2003.51(10): p.2837-
2853.
4.Lerner A. B., Fitzpatrick T. B., Calkins E., Summerson
W. H. Mammalian tyrosinase : preparation and properties.
J Biol Chem,1949.p.185-195.
5.Korner, A., Pawelek J. Mammalian tyrosinase catalyzes
three reactions in the biosynthesis of melanin. Science,
1982.217(4565):p.1163-1165.
6.Wang N., Hebert D. N. Tyrosinase maturation through the
mammalian secretory pathway: bringing color to life.
Pigment Cell Res,2006.19(1):p.3-18.
7.Land E. J., Ramsden C. A., Riley P. A. Tyrosinase
autoactivation and the chemistry of ortho-quinone
amines. Acc Chem Res,2003.36(5):p.300-308.
8.Curto E. V., Kwong C., Hermersdorfer H., Glatt H.,
Santis C., Virador V., Hearing V. J. Jr., Dooley T. P.
Inhibitors of mammalian melanocyte tyrosinase: in vitro
comparisons of alkyl esters of gentisic acid with other
putative inhibitors. Biochem Pharmacol,1999.57(6):p.663-
672.
9.Niwa Y., Akamatsu H. Kojic acid scavenges free radicals
while potentiating leukocyte functions including free
radical generation. Inflammation,1991.15(4):p.303-315.
10.Hammond C., Braakman I., Helenius A. Role of N-linked
oligosaccharide recognition, glucose trimming, and
calnexin in glycoprotein folding and quality control.
Proc Natl Acad Sci U S A,1994.91(3):p.913-917.
11.Ware F. E, Vassilakos A., Peterson P. A, Jackson M. R,
Lehrman M. A, Williams D. B. The molecular chaperone
calnexin binds Glc1Man9GlcNAc2 oligosaccharide as an
initial step in recognizing unfolded glycoproteins. J
Biol Chem,1995.270(9):p.4697-4704.
12.Olivares, C., Solano, F. Garcia-Borro J.C. Conformation-
dependent post-translational glycosylation of
tyrosinase. Requirement of a specific interaction
involving the CuB metal binding site. J Biol Chem,2003.
278(18):p.15735-15743.
13.Wang Y., Androlewicz M. J. Oligosaccharide trimming
plays a role in the endoplasmic reticulum-associated
degradation of tyrosinase. Biochem Biophys Res Commun,
2000.271(1):p.22-27.
14.Svedine, S., Wang T., Halaban R., Hebert D. N.
Carbohydrates act as sorting determinants in ER-
associated degradation of tyrosinase. J Cell Sci,2004.
117(14):p.2937-2949.
15.Arvan P., Zhao X., Ramos-Castaneda J., Chang A.
Secretory pathway quality control operating in Golgi,
plasmalemmal, and endosomal systems. Traffic,2002.3(11):
p.771-780.
16.Ando H.,Kondoh H., Ichihashi M. Hearing V.J.Approaches
to identify inhibitors of melanin biosynthesis via the
quality control of tyrosinase. J Invest Dermatol,2007.
127(4):p.751-761.
17.Tachibana M., Takeda K., Nobukuni Y., Urabe K., Long J.
E., Meyets K. A. Ectopic expression of MITF, a gene for
Waardenburg syndrome type 2, converts fibroblasts to
cells with melanocyte characteristics. Nat Genet,1996.14
(1):p.50-54.
18.Imokawa G., Mishima Y. Loss of melanogenic properties
in tyrosinases induced by glucosylation inhibitors
within malignant melanoma cells. Cancer Res,1982.42
(5):p.1994-2002.
19.Maeda, K., Fukuda M. Arbutin: mechanism of depigmenting
action in human melanocyte culture. J Pharmacol Exp
Ther,1996.276(2):p.765-769.
20.Mishima Y., Hatta S., Ohyama Y., Inazu M. Induction of
melanogenesis suppression: cellular pharmacology and
mode of differential action. Pigment Cell Res,1988.1
(6):p.367-374.
21.Funasaka Y., Chakrabory A. K., Komoto M., Ichihashi M.
The depigmenting effect of alpha-tocopheryl ferulate on
human melanoma cells. Br J Dermatol,1999.141(1):p.20-29.
22.Kumano Y., Sakamoto T., Egawa M., Lwai I., Tanaka M.,
Yamonoto I. In vitro and in vivo prolonged biological
activities of novel vitamin C derivative, 2-O-alpha-D-
glucopyranosyl-L-ascorbic acid (AA-2G), in cosmetic
fields. J Nutr Sci Vitaminol (Tokyo),1998.44(3):p.345-
359.
23.Ando H., Wen Z. M., Kim H. Y., Valencia J. C.
Intracellular composition of fatty acid affects the
processing and function of tyrosinase through the
ubiquitin-proteasome pathway. Biochem. J.2006.p.43-50.
24.吳政億. 蘭嶼山欖葉部成分之研究. 臺北醫學大學生藥學研究所
碩士論文,2005.
25.Masataka S. and Kikuchi M. Phenolic glycosides from
Osmanthus asiaticus. Phytochemistry,1991.30(9):p.3147-
3149.
26.黃俐瑋. 蘭嶼肉桂葉部成分之研究. 臺北醫學大學生藥學研究
所碩士論文,2005.
27.陳克君. 金絲草成分之研究. 臺北醫學大學生藥學研究所碩士論
文,2006.
28.林怡沛. 台灣蘋果及雞血藤之抗氧化活性成分探討. 臺北醫學大
學生藥學研究所碩士論文,2005.
29.Duan, H., Zhang Y., Xu J., Qiao J., Suo Z., Mu X.
Effect of anemonin on NO, ET-1 and ICAM-1 production in
rat intestinal microvascular endothelial cells. J
Ethnopharmacol,2006.104(3):p.362-366.
30.Zhou Y., Hu S. Hu S., Liang G. Study on peparation of
anemonin from Ranunculus japonicus Thunb. Journal of
Chinese medicinal materials,2004.27(10):p.762-764.
31.Kern J. R., Cardellina J. H. Native American medicinal
plants. Anemonin from the horse stimulant Clematis
hirsutissima. J Ethnopharmacol,1983.8(1):p.121-123.
32.Dong C. X., Shi S. P., Wu K. S., Tu P. F. Studies on
chemical constituents from root of Clematis hexapetala.
China journal of Chinese materia media,2006.31
(20):p.1696-1699.
33.He M., Z J., Hu C. Studies on the Chemical Compounds of
Clematis chinensis. Journal of Chinese Pharmaceutical
Sciences,2001.10(4):p.180-182.
34.Hill R., Van H. R. Ranunculin; the precursor of the
vesicant substance of the buttercup. Biochem J,1951.49
(3): p.332-335.
35.Noboru S, Hiroshi K., Kazutosi Y. The synthesis of
anemonin by the photochemical dimerization of
protoanemonin. Yukei Gosei Kagaku Kyokai Shi,1967.25
(7):p.582.
36.杜立春, 胡., 白頭翁素的合成. 中國醫藥工業雜誌,2001.32
(5):p.201-202.
37.Ning Y. M., Rao Y. F., Liang W. Q. Influence of
permeation enhancers on transdermal permeation of
anemonin. China Journal of Chinese Materia
Medica,2007.22(5):p.393-396.
38.Martin M. L., Ortiz d. U. A. V., Montero M. J., Carron
R., San R. L. Pharmacologic effects of lactones
isolated from Pulsatilla alpina subsp. apiifolia. J
Ethnopharmacol, 1988.24(2-3):p.185-191.
39.謝珮文, 石竹科植物菁芳草、瞿麥、長萼瞿麥化學成分及
Dianthramide類生物鹼化合物之合成與其生物活性之研究. 高雄
醫學大學藥學研究所博士論文,2004.
40.Ning Y. M., Liu X. Y., Liang W. Q. Preparetion of
anemonin dermal patches and its transdermal permeation
in vitro. Herald of Medical.2006.25(11):p.1121-1123.
41.Mosmann T. Rapid colorimetric assay for cellular growth
and survival: application to proliferation and
cytotoxicity assays. J Immunol Methods,1983.65(1-2):p.
55-63.
42.Smith P. K., Kroha R. I., Hermanson G. T., Mallia A.
K., Gartner F. H., Provenzano M. D., Fujimoto E. K.,
Goeke N. M., Olson B. J., Klenk D. C. Measurement of
protein using bicinchoninic acid. Anal Biochem,1985.150
(1):p.76-85.
43.Kushimoto T., Basrur V., Valencia J., Matsunaga J.,
Vieira W. D., Ferrans V. J., Muller J., Appella E.,
Hearing V. J. A model for melanosome biogenesis based
on the purification and analysis of early melanosomes.
Proc Natl Acad Sci USA,2001.98(19):p.10698-10703.
44.Hearing V. J., Tsukamoto K. Enzymatic control of
pigmentation in mammals. Faseb J,1991.5(14):p.2902-2909.
45.Min Kyung Cho, Young P. J., Young C. K., Sang G. K.
Arctigenin, a phenylpropanoid dibenzylbutyrolactone
lignan, inhibits MAP kinases and AP-1 activation via
potent MKK inhibition: the role in TNF-α inhibition.
Int Immunopharmacol,2004.4(10-11):p.1419-1429.
46.Yanase, H., Ando H., Horikawa M., Watanabe M., Mori T.,
Matsuda N. Possible Involvement of ERK 1/2 in UVA-
Induced Melanogenesis in Cultured Normal Human
Epidermal Melanocytes. Pigment Cell Research,2001.14
(2):p.103-109.
47.Kim D. S., Hwang E. S., Lee J. E., Kim S. Y., Park K. C. Sphingosine-1-phosphate promotes mouse melanocyte
survival via ERK and Akt activation. Cellular
Signalling, 2003.15(10):p.919-926.
48.Martin, M. L., Oetiz Don. U. A. V. Montero M. J.,
Carrron R., San R. L. Pharmacologic effects of lactones
isolated from Pulsatilla alpina subsp. apiifolia. J
Ethnopharmacol,1988.24(2-3):p.185-191.
49.Sestakova, B., Vachtenheim J. Distinct co-regulation of
endogenous versus transfected MITF-dependent tyrosinase
promoter. Folia Biol.2006.52(5):p.161-166.
50.Bentley N. J., Eisen T., Goding C. R. Melanocyte-
specific expression of the human tyrosinase promoter:
activation by the microphthalmia gene product and role
of the initiator. Mol Cell Biol,1994.14(12):p.7996-8006.
51.Jiao, Z., Mollanyakaghababa R., Pavan W. J., Antonellis
A., Green E. D.,Direct interaction of Sox10 with the
promoter of murine Dopachrome Tautomerase (Dct) and
synergistic activation of Dct expression with Mitf.
Pigment Cell Res,2004.17(4):p.352-362.
52.Busca, R. Ballotti R. Cyclic AMP a Key Messenger in the
Regulation of Skin Pigmentation. Pigment Cell Research,
2000.13(2):p.60-69.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關期刊