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研究生:唐婉綺
研究生(外文):Tang,Wan-Chi
論文名稱:重組人類酪胺酸酶抑制劑篩選及其對黑色素細胞之影響
論文名稱(外文):Selection of Recombinant Human Tyrosinase Inhibitors and Their Effect on Melanocyte
指導教授:江善宗陳俊宏陳俊宏引用關係
指導教授(外文):Jiang,Shann-TzongChen,Gen-Hung
口試委員:江善宗陳俊宏許成光
口試委員(外文):Jiang,Shann-TzongChen,Gen-HungSyu,Cheng-Guang
口試日期:2015-06-17
學位類別:碩士
校院名稱:靜宜大學
系所名稱:化粧品科學系
學門:民生學門
學類:美容學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:116
中文關鍵詞:酪胺酸酶Pichia pastoris GS115黑色素6種天然豆類萃取液綠豆小田豆黑豆黃豆石斛酚益母草鹼羥基化白藜蘆醇
外文關鍵詞:tyrosinase(RHT)Pichia pastoris GS115 expression system16 natural pulse extractsmung beanbroad beenblack beanyellow beendendrophenolleonurine hydrochloridehydroxyresveratrol
相關次數:
  • 被引用被引用:1
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  • 下載下載:14
  • 收藏至我的研究室書目清單書目收藏:0
黑色素(Melanin)普遍存在於生物,如微生物、植物、動物等,它主要提供人體極其重要的生理功能,其通過吸收UV光防止紫外線(UV)損害皮膚,避免造成皮膚癌。然而,黑色素合成開始第一步關鍵酶為酪胺酸酶(Tyrosinase,TYR),它是一個極為結構複雜且含銅的酵素,不過如果它分泌太多時,在人體上會產生色素沉著性疾病,如雀班等,因此以重組人類酪胺酸酶及黑色素為指標做為抑制劑之應用。本研究有三個階段,分別為構築重組人類酪胺酸於表現載體且轉殖進入酵母菌、重組人類酪胺酸於大腸桿菌內涵體表現和抑制劑篩選及純物質對黑色素影響。
重組人酪胺酸酶(recombinant human tyrosinase, RHT)已在我們的實驗室被選殖,高量表現成包涵體和並復性為活性形態。本研究以此重組人類酪胺酸酶當指標從16種天然豆類萃取液中分析是否有抑制劑或基質之成分。此外,在我們的實驗室先前之實驗中,篩選自十幾種植物萃,並以動力學分析後具RHT抑制活性的關鍵成分標準品,石斛酚、益母草鹼和羥基化白藜蘆醇,在本研究中已進一步以B16F10黑色素細胞進行研究已探索其對黑色素生成之影響。根據這些結果,這三個純化合物顯示在B16F10黑素細胞中有抑制能力。從豆類萃取液體中,綠豆、小田豆、黑豆和黃豆有顯著抑制RHT活性之效果。

Melanin is ubiquitously present in organisms, as bacteria, plants, and animals. It provides a variety of functions and serves human body extremely important physiological functions, which possess preventing ultraviolet (UV) damage by absorbing UV light and then protect the skin to avoid causing the skin cancer.(Chiang et al., 2014) Melanin synthesis is initiated with the first step of the tyrosinase, copper–containing enzyme. In contrast, melanin is excessive secretion of melanin pigments cause cumulative of melanin in the skin and then lead to skin disorders such as age spots, freckles, melasma and malignant melanoma. The study has three points: (1)cloning the recombinant human tyrosinase (RHT) into Pichia pastoris GS115 expression system, (2)Selection of the RHT inhibitors from 16 natural pulse extracts, (3)Evaluating the effect of 3 pure compounds on melanocyte.
Recombinant human tyrosinase (RHT) had been cloned, over-expressed in inclusion body and renatured into active form before in our laboratory, and was used as a probe in this study for searching the natural inhibitors or substrates from 16 natural pulse extracts. Furthermore, 3 pure compounds, dendrophenol, leonurine hydrochloride and hydroxyresveratrol, which were the critical constituents from of three different plants selected from dozens of plants in the experiments before in our laboratory and exhibited kinetically inhibitory activity, evaluated before by the same RHT, were further investigated their effect on melanization using B16F10 melanocyte. According to these results, the three pure compound exhibited inhibitory ability of melanin synthesis in B16F10 melanocyte; and the extracts from mung bean, broad been, black bean and yellow been showed significantly RHT inhibitory activity.

摘要I
英文摘要III
目錄V
表目錄VIII
圖目錄X
第一章、前言1
第二章、文獻回顧3
第一節、皮膚結構3
第二節、皮膚色素6
第三節 、黑色素生成過程9
1-3-1黑色素細胞(melanocyte)9
1-3-2黑色素生成機制10
1-3-3黑色素的功能15
第四節、酪胺酸酶介紹及作用19
1-4-1 酪胺酸酶簡介19
1-4-2活性中心的結構21
第五節、黑色素生成抑制方法23
1-5-1酪胺酸酶活性或黑色素生成抑制方法23
第六節、豆類及純物質介紹26
1-6-1豆類介紹26
第三章、實驗設計30
第四章、實驗材料與方法31
第一節、實驗材料31
第二節、實驗方法34
第五章、結果與討論46
5-1重組人類酪胺酸酶基因與載體之構築46
5-2重組 the human tyrosinase-pPIC9 Vector制備與轉殖於酵母菌47
5-3 the human tyrosinase- pET23a(+) Vector轉殖於Ecoli、蛋白質表現及電泳分析48
5-4人類酪胺酸酶復性及活性測定49
5-5十六種豆類萃取液對人類酪胺酸酶篩選50
5-6不同濃度豆類萃取液對人類酪胺酸酶活性變化52
5-7萃取液抑制劑的Lineweaver-Burk plot作圖54
5-8檢品對於 (B16-F10)細胞存活率測定55
5-8-1石斛酚(dendrophenol) 對於 (B16-F10)細胞存活率測定55
5-9石斛酚(dendrophenol) 對於細胞黑色素含量測定56
5-10益母草鹼(Leonurine hydrochloride)對於 (B16-F10)細胞存活率測定57
5-11益母草鹼(Leonurine hydrochloride)對於細胞黑色素含量測定58
5-12 羥基白藜蘆醇(Oxyresveratrol)對於 (B16-F10)細胞存活率測定59
5-13羥基白藜蘆醇(Oxyresveratrol) 對於細胞黑色素含量測定60
第六章結論61
第七章、參考文獻110
表一、黑豆萃取物在不同濃度基質對重組酪胺酸酶之動力學參數及抑制型態92
表二、不同濃度甲醇對於 (B16-F10)細胞存活率測定93
表三、石斛酚(dendrophenol) 對於 (B16-F10)細胞存活率測定94
表四、石斛酚(dendrophenol) 對於 (B16-F10)細胞存活率測定95
表五、石斛酚(dendrophenol) 對於細胞黑色素含量測定(細胞數目以細胞存活率計算)96
表六、石斛酚(dendrophenol) 對於細胞黑色素含量測定(細胞數目以細胞存活率計算)97
表七、石斛酚(dendrophenol) 對於細胞黑色素含量測定(細胞數目以蛋白質吸光值計算)98
表八、石斛酚(dendrophenol) 對於細胞黑色素含量測定(細胞數目以蛋白質吸光值計算)98
表九、石斛酚(dendrophenol) 對於細胞黑色素含量測定100
表十、石斛酚(dendrophenol) 對於細胞黑色素含量測定101
表十一、益母草鹼(Leonurine hydrochloride)對於 (B16-F10)細胞存活率測定102
表十二、益母草鹼(Leonurine hydrochloride) 對於細胞黑色素含量測定(細胞數目以蛋白質吸光値計算)103
表十三、益母草鹼(Leonurine hydrochloride) 對於細胞黑色素含量測定(細胞數目以細胞純活率值計算)104
表十四、益母草鹼(Leonurine hydrochloride) 對於細胞黑色素含量測定105
表十五、羥基白藜蘆醇(Oxyresveratrol)對於 (B16-F10)細胞存活率測定106
表十六、羥基白藜蘆醇(Oxyresveratrol)對於 (B16-F10) 對於細胞黑色素含量測定(細胞數目以蛋白質吸光值計算)107
表十七、羥基白藜蘆醇(Oxyresveratrol)對於 (B16-F10) 對於細胞黑色素含量測定(細胞數目以細胞存活率計算)108
表十八、羥基白藜蘆醇(Oxyresveratrol)對於 (B16-F10) 對於細胞黑色素含量測定109
附圖一-表皮層5
附圖二-皮膚的結構6
附圖三- 人類色素沉著,主要膚質:非裔美國人,亞裔,白人,西班牙裔和(從左至右)8
附圖四-在黑色素的生物合成早期階段11
附圖五 -黑色素的示意圖13
附圖六-黑色素細胞在基底膜的皮層上合成黑色素於黑色素體內,其藉由胞吐機制被排出囊泡到角質細胞15
附圖七-雙孢菇酪胺酸酶之雙核銅結合位點的幾何圖形20
附圖八-催化循環為由氧存在下酪胺酸酶monophenol氧化和diphenol基質到o-quinones 22
圖一、重組人類tyrosinase 基因之colony PCR 產物之洋菜膠電泳分析圖64
圖二、重組人類tyrosinase 基因之限制酶及PCR產物之洋菜膠電泳分析圖65
圖三、pPIC9 -重組人類tyrosinase vector 之 DNA 定序圖譜(primer)66
圖四、重組人類tyrosinase –pET23a(+)表現載體之PCR產物電泳分析圖67
圖五、純化的重組 human tyrosinase之 SDS-PAGE 電泳分析圖68
圖六、酵素活性測定作圖69
圖七、赤碗豆及加拿大黃碗豆對重組酪胺酸酶篩選測定作圖70
圖八、美國碗豆、澳洲Dunpeas及加拿大碗豆對重組酪胺酸酶篩選測定作圖71
圖九、紅豆及綠豆對重組酪胺酸酶篩選測定作圖72
圖十、小田豆、雪蓮子及花豆對重組酪胺酸酶篩選測定作圖73
圖十一、加拿大黃豆及美白豆對重組酪胺酸酶篩選測定作圖74
圖十二、黑豆及黃豆對重組酪胺酸酶篩選測定作圖75
圖十三、不同濃度赤碗豆萃取物對重組酪胺酸酶測定作圖76
圖十四、不同濃度加拿大黃碗豆萃取物對重組酪胺酸酶測定作圖77
圖十五、不同濃度美國碗豆萃取物對重組酪胺酸酶測定作圖78
圖十六、不同濃度澳洲Dunpeas萃取物對重組酪胺酸酶測定作圖79
圖十七、不同濃度加拿大4010豆萃取物對重組酪胺酸酶測定作圖80
圖十八、不同濃度楓豆萃取物對重組酪胺酸酶測定作圖81
圖十九、不同濃度綠豆萃取物對重組酪胺酸酶測定作圖82
圖二十、不同濃度小田豆萃取物對重組酪胺酸酶測定作圖83
圖二十一、不同濃度雪蓮子萃取物對重組酪胺酸酶測定作圖84
圖二十二、花豆萃取物對重組酪胺酸酶測定作圖85
圖二十三、不同濃度小野豆萃取物對重組酪胺酸酶測定作圖86
圖二十四、不同濃度加拿大黃豆萃取物對重組酪胺酸酶測定作圖87
圖二十五、不同濃度美白豆萃取物對重組酪胺酸酶測定作圖88
圖二十六、不同濃度黑豆萃取物對重組酪胺酸酶測定作圖89
圖二十七、不同濃度黃豆萃取物對重組酪胺酸酶測定作圖90
圖二十八、黑豆萃取物與基質對重組酪胺酸酶之Lineweaver-Burk作圖 91
圖二十九、不同濃度甲醇對於 (B16-F10)細胞存活率測定93
圖三十、石斛酚(dendrophenol) 對於 (B16-F10)細胞存活率測定94
圖三十一、石斛酚(dendrophenol) 對於 (B16-F10)細胞存活率測定95
圖三十二、石斛酚(dendrophenol) 對於細胞黑色素含量測定101
圖三十三、益母草鹼(Leonurine hydrochloride)對於 (B16-F10)細胞存活率測定102
圖三十四、益母草鹼(Leonurine hydrochloride)對於細胞黑色素含量測定105
圖三十五、羥基白藜蘆醇(Oxyresveratrol)對於 (B16-F10)細胞存活率測定106
圖三十六、羥基白藜蘆醇(Oxyresveratrol)對於細胞黑色素含量測定109
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