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研究生:林育民
研究生(外文):Yu-MIn Lin
論文名稱:Ectoine 對黑色素瘤細胞的黑色素生成之影響探討研究
論文名稱(外文):Effect of ectoine on the melanogenesis of B16-F0 and A2058 cell lines
指導教授:姚少凌魏毓宏朱一民朱一民引用關係黃效民黃效民引用關係
指導教授(外文):Chao-Ling YaoYu-Hong WeiI-Ming Chu
學位類別:碩士
校院名稱:元智大學
系所名稱:化學工程與材料科學學系
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:65
中文關鍵詞:酪胺酸?美白ectoine
外文關鍵詞:tyrosinasewhiteningectoine
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Ectoine是一種存在嗜鹽性微生物內發現的一種生物分子,其作用在於保護細菌以利存活在高滲透壓以及一些極端惡劣的環境,如脫水、加熱、冷凍和乾燥等。目前有文獻指出Ectoine具有防曬、抗老化、保持皮膚彈性、抗氧化、抗UV等多種功效,然而卻只有少數美白能力相關的報導。
因此在本研究中,以老鼠黑色素瘤細胞株(B16-F0)與人類黑色素瘤細胞株(A2058)做為測試平台,測試Ectoine是否具有美白的能力。實驗結果顯示,在不影響細胞存活率的前提下,細胞內的黑色素含量明顯地受到Ectoine所抑制,在於B16-F0細胞株與A2058細胞株之下其IC50值分別為 156.50 μM和432.42 μM。在於酪胺酸?的活性抑制效果上,皆與兩株細胞株接觸的Ectoine濃度成正比分別為 879.22 μM和470.80 μM。而在Real-time PCR實驗中,發現了對於B16-F0細胞株,其中的酪胺酸?、TRP1、TRP2與MITF的表現皆會受到Ectoine所影響。而A2058細胞株中,卻只有TRP2與MITF的表現會受到Ectoine所影響。在於蛋白質表現上,這兩株細胞株對於酪胺酸?、TRP1、TRP2與MITF的表現皆有影響。基於目前的實驗結果確定了Ectoine具有美白的能力。


In order to search new agents with a depigmenting effect, we have found Ectoine derived from halophilic microorganism. The role of ectoine has been studied extensively with regard to not only the protection of cells from high external osmolarity but also the stabilization of biomolecules in cells against dehydration, heating, freezing or drying. Now many reports claimed that Ectoine has variey of biological activities, including sunscreen, anti-aging, maintenance of skin elasticity, antioxidant and anti-UV.
In this report, we studied ectoine on its function in the skin-whitening area. Firstly, we selected mouse melanoma cell line(B16-F0)and human melanoma cell line(A2058)as testing cells in all of our experiments. The ectoine showed low cytotoxicity even at 500 μM. Secondly, we evaluated melanogenesis effect and tyrosinase relative activity by treating melanoma cells with different concentration of ectoine. The ectoine inhibited melanin formation in B16-F0 and A2058 melanoma cell lines with IC50 values of 156.50 and 432.42 μM, respectively. In addition, ectoine displayed a significant inhibitory effect on cellular and mushroom tyrosinase activity. The analysis of inhibitory kinetics revealed that ectoine acted a noncompetitive inhibitory effect on tyrosinase. Importantly, we found that the melanin content and tyrosinase activity were decreased obviously with increasing concentration of ectoine. In the Real-time PCR experiments,we found that the B16-F0 cell line, tyrosinase, TRP1, TRP2 and the MITF will be Ectoine are affected. In the A2058 cell line, but only TRP2 and MITF will be affected by Ectoine. We found protein expression of cells were decreased obviously with increasing concentration of ectoine.


目錄
摘 要 III
ABSTRACT IV
目錄 VI
圖目錄 X
表目錄 XII
中英文對照表 XIII
1.1 前言 1
1.2 研究動機 3
1.3 Ectoine介紹 4
1.4 皮膚構造 5
1.4.1 表皮組織(epidermis) 6
1.4.2 真皮層(dermis) 8
1.4.3 皮下組織(subcutaneous layer) 8
1.5 黑色素生成路徑 9
1.6 生成黑色素重要蛋白質 13
1.6.1 酪胺酸? 13
1.6.2 TRP-1 13
1.6.3 TRP-2 14
1.7 抑制黑色素生成的機制 14
1.8 常見美白藥物 15
1.8.1熊果素 15
1.8.2麴酸 16
1.8.3鞣花酸 16
1.8.4對苯二酚與汞 17
1.9 酵素活性的抑制類型 19
1.9.1 競爭型抑制(competitive inhibition) 20
1.9.2 非競爭型抑制(non-competitive inhibition) 20
1.9.3 反競爭型抑制(un-competitive inhibition) 20
第二章 實驗材料與方法 22
2.1 實驗材料 22
2.1.1 細胞 22
2.1.2 藥品 22
2.1.3 使用儀器 24
2.2 實驗方法 24
2.2.1 B16-F0與A2058細胞培養 24
2.2.2 黑色素定量曲線 25
2.2.3黑色素瘤細胞內黑色素測試 26
2.2.4 黑色素瘤細胞存活率測試 27
2.2.5黑色素瘤細胞內酪胺酸?活性測試 28
2.2.5 菇類酪胺酸?活性測試 29
2.2.6 Lineweaver-Burk plots 30
2.2.7 即時定量PCR(Quantitative Real-time PCR, qPCR) 31
2.2.8 西方墨點法(Western Blot) 32
第三章 實驗結果與討論 38
3.1 Ectoine對黑色素瘤細胞存活率影響 38
3.2 Ectoine對黑色素瘤細胞內黑色素含量影響 41
3.3 Ectoine對菇類酪胺酸?活性影響 44
3.4 Ectoine對細胞內酪胺酸?活性影響 45
3.5 Ectoine對酪胺酸?活性抑制機制 47
3.6 Ectoine對黑色素瘤細胞內基因之影響 49
3.7 Ectoine對形成黑色素之蛋白質表現影響 55
第四章 結果與討論 57
第五章 未來展望 59
第六章 參考文獻 60



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