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研究生:唐郁評
研究生(外文):Yu-Ping Tang
論文名稱:探討香椿萃取物抑制黑色素生成之分子機制
論文名稱(外文):Effect of Toona sinensis extracts on molecular mechanism of anti-melanogenesis
指導教授:張素瓊張素瓊引用關係
指導教授(外文):Sue-Joan Chang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生命科學系碩博士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:109
中文關鍵詞:黑色素香椿美白黑色素小體轉移酪胺酸��
外文關鍵詞:Toona sinensiswhiteningmelanosome transfermelanintyrosinase
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近年來,臭氧層受到破壞,導致陽光中的紫外線更加強烈,促使黑色素生成加快,降低皮膚上的黑色素生成成為化妝/保養品的主力,但化學性化妝/保養品對於皮膚具有較強的刺激性而造成使用上的困擾,故尋找有效天然不具毒性之黑色素生成抑制劑乃蔚為風潮。文獻記載香椿 ( Toona sinensis ) 全樹各部分均有保健或治療之功能。近年研究發現,香椿中主要的抗氧化成分為槲黃素 ( Quercetin ) 及�坉馱l酸 ( Gallic acid ),具有良好抗氧化能力。本研究主要探討不同香椿萃取物 ( TS ) 對小鼠黑色素細胞株 ( B16-F10 ) 抑制黑色素生成與相關之分子機制、抑制黑色素小體從黑色素細胞株轉移到角質細胞株 ( XB-2 ) 之機制、對皮膚角質細胞生長之影響。分別處理細胞 DMEM 培養液 ( 控制組 )、熊果素 ( Arbutin )、槲黃素 ( Quercetin )、�坉馱l酸 ( Gallic acid )、香椿萃取物TSL-2、TSL-2P及TSL-6等七組。測定細胞存活實驗 ( MTT )、黑色素 ( Melanin ) 生成、酪胺酸�� ( Tyrosinase ) 活性、B16-F10細胞內 Tyrosinase、TRP-1、TRP-2及MITF 等黑色素生成路徑相關酵素之基因變化及蛋白質表現、MITF在B16-F10細胞中之位置變化與黑色素小體轉移實驗等。
實驗結果發現不同香椿萃取物 ( TSL-2、TSL-2P及TSL-6 ) 對於細胞內黑色素生成與酪胺酸�◆簿嶼〝坌狾釦磻謜蘆G。為了進一步瞭解作用機制,利用反轉錄聚合��鏈鎖反應 ( RT-PCR ) 與西方墨點法 ( Western blotting ),分析B16-F10細胞內 Tyrosinase、TRP-1、TRP-2及MITF 等黑色素生成路徑相關酵素的基因變化及蛋白質表現差異,發現香椿萃取物 ( TSL-2P與TSL-6 ) 可抑制Tyrosinase與TRP-2基因表現,香椿萃取物 ( TSL-2P與TSL-6 ) 可抑制Tyrosinase、TRP-1與TRP-2蛋白表現;MITF為調控黑色素生成之上游蛋白,平常會表現在細胞質與細胞核中,當細胞活化產生黑色素時,細胞質中的MITF會向細胞核移動,本研究利用免疫細胞化學染色法 ( Immunocytochemistry ) 探討不同處理對MITF在B16-F10細胞中之位置變化,發現香椿萃取物 ( TSL-6 ) 可減少MITF在細胞核中表現,進而抑制下游相關酵素表現。利用Co-incubation實驗,將B16-F10小鼠黑色素細胞與XB-2小鼠角質細胞依照1比10之比例一起培養,利用免疫細胞化學染色法與流式細胞儀 ( Flow cytometer ) 觀察其抑制黑色素小體轉移的現象,發現香椿萃取物 ( TSL-6 ) 可有效抑制黑色素小體轉移。最後,利用細胞增生實驗發現三種香椿萃取物皆無法加速角質細胞生長。
綜合以上結果發現,TSL-6為三種香椿萃取物中最有效抑制黑色素生成的萃取物,且TSL-6可經由許多機制來調控抑制黑色素的生成:(1) 抑制酪胺酸�� ( 黑色素生成速率決定酵素 ) 的活性,(2) 抑制酪胺酸�﹛BTRP-1與TRP-2蛋白質表現,(3) 抑制酪胺酸�◆PTRP-2基因表現,(4) 減少MITF在細胞核中表現,(5) 抑制黑色素小體轉移。此結果顯示香椿萃取物TSL-6可成為新穎美白的潛力產品,或是提昇市售既有產品美白的功能。
Recently, the melanin synthesis is increased on the skin due to the destruction of ozone layer resulting in the elevated intensity of ultraviolet from the sunlight. To reduce skin melanin synthesis become popular cosmetics/ skin care products. However, the chemical-based cosmetics/ skin care products usually have side effects such as irritation, or rash. Therefore the effective natural products of melanin inhibitors are well accepted by the consumer. According to the literature, Toona sinensis (TS) of various parts has many functions for health care and the treatment of disorders. Recent studies indicated that the main anti-oxidant compounds in Toona sinensis were quercetin and gallic acid. In this study, cell lines were treated with different Toona sinensis extracts, to investigated the molecular mechanism of melanin synthesis inhibition using murine melanoma cell line (B16-F10), the effect on melanosome transfer from melanocytes to keratinocytes (XB-2), and the effect on inducton of keratinocytes growth. The cell viability (MTT), melanin synthesis, tyrosinase activity, gene and protein expression of tyrosinase, TRP-1, TRP-2 and MITF related of melanin synthesis pathway, localization of MITF in B16-F10 cell lines and melanosome transfer from melanocytes to keratinocytes were deteced in B16-F10 treated with DMEM medium ( control ), arbutin, quercetin, gallic acid, TSL-2, TSL-2P and TSL-6.
Our results showed that Toona sinensis extracts inhibited the cellular melanin synthesis and tyrosinase activity. The gene and protein expression were analyzed by RT-PCR and western blotting of tyrosinase, TRP-1, TRP-2 and MITF involving in melanin synthesis pathway. We found that Toona sinensis extracts ( TSL-2P and TSL-6 ) reduced gene expression of tyrosinase and TRP-2, and reduced protein expression of tyrosinase, TRP-1 and TRP-2. MITF is the up-stream protein of melanogenesis, we used immunocytochemistry to detect the MITF protein localization in B16-F10 cell line. And found that Toona sinensis extracts ( TSL-6 ) reduced the translocation of MITF in the nucleus, thereby inhibiting the transcription of downstream protein. The B16-F10 ( melanocytes ) and XB-2 ( keratinocytes ) cell lines were co-culture to observe the inhibition of melanosome transfer by using immunocytochemistry and flow cytometry. We found that Toona sinensis extracts ( TSL-6 ) inhibited melanosome transfer efficiently. However, all three extracts of Toona sinensis did not induce keratinocytes growth using MTT assay.
In conclution, TSL-6 is the most effective one among three extracts of Toona sinensis for inhibition of melanin generation. The mechanism by which TSL-6 regulates the inhibition of melanin synthesis include: (1) inhibiting tyrosinase enzyme ( rate-limiting enzyme of melanin synthesis ) activity, (2) inhibiting the protein expression of tyrosinease, TRP-1 and TRP-2, (3) inhibiting the gene expression of tyrosinase and TRP-2, (4) reducing MITF expression in the nucleus, (5) inhibiting melanosome transfer. These results suggested that the Toona sinensis extracts TSL-6 is potential to develop into new whitening cosmetics, or enhance the feasibility of the existing whitening product.
中文摘要----------------------------------------------------------------------------- I
英文摘要----------------------------------------------------------------------------- III
致謝--------------------------------------------------------------------------------- V
目錄--------------------------------------------------------------------------------- VII
表目錄------------------------------------------------------------------------------- X
圖目錄------------------------------------------------------------------------------- XI
第一章:前言------------------------------------------------------------------------- 1
第二章:文獻探討--------------------------------------------------------------------- 3
1.美白觀念------------------------------------------------------------------------- 3
2.皮膚簡介------------------------------------------------------------------------- 6
3.黑色素細胞簡介------------------------------------------------------------------- 7
2.1黑色素小體-------------------------------------------------------------------- 8
2.2黑色素------------------------------------------------------------------------ 9
2.3黑色素生成及調控-------------------------------------------------------------- 10
4.酪胺酸?簡介--------------------------------------------------------------------- 12
4.1角質細胞調控MITF活化---------------------------------------------------------- 13
4.2經後轉譯作用抑制MITF之相關調控路徑-------------------------------------------- 15
5.角質細胞簡介--------------------------------------------------------------------- 16
6.美白調控------------------------------------------------------------------------- 17
6.1阻斷皮膚黑色素生成的酵素活性-------------------------------------------------- 17
6.2降低黑色素小體轉移到角質細胞-------------------------------------------------- 18
6.3加速皮膚的汰換---------------------------------------------------------------- 18
7.抗氧化與美白--------------------------------------------------------------------- 19
8.中草藥與美白--------------------------------------------------------------------- 19
9.香椿----------------------------------------------------------------------------- 20
9.1國內外香椿研究成果------------------------------------------------------------ 22
第三章:材料與方法------------------------------------------------------------------- 24
實驗藥品--------------------------------------------------------------------------- 24
實驗儀器--------------------------------------------------------------------------- 27
常用容易配置----------------------------------------------------------------------- 28
實驗架構--------------------------------------------------------------------------- 30
香椿萃取流程----------------------------------------------------------------------- 31
實驗方法--------------------------------------------------------------------------- 32
第四章:結果------------------------------------------------------------------------- 39
1.不同濃度之處理物對B16-F10黑色素細胞株存活的影響---------------------------------- 39
2.不同濃度之處理物對XB-2角質細胞株存活的影響--------------------------------------- 40
3.不同濃度之處理物對B16-F10黑色素細胞株形態的影響---------------------------------- 40
4.不同濃度之處理物對B16-F10黑色素細胞株抑制黑色素生成的影響------------------------ 40
5.不同濃度之處理物對B16-F10黑色素細胞株抑制酪胺酸?活性的影響---------------------- 41
6.不同處理物對B16-F10黑色素細胞株中黑色素生成路徑相關酵素的蛋白質表現差異的影響---- 41
7.不同處理物對B16-F10黑色素細胞株中黑色素生成路徑相關酵素的基因表現差異的影響------ 42
8.不同濃度之處理物對B16-F10黑色素細胞株MITF蛋白位置的影響-------------------------- 43
9.不同處理物對B16-F10黑色素細胞株與XB-2角質細胞Co-culture之黑色素小體轉移的影響---- 44
10.不同處理物對XB-2角質細胞株增生的影響-------------------------------------------- 45
第五章:討論------------------------------------------------------------------------- 46
1.不同濃度之處理物對B16-F10黑色素細胞株與XB-2角質細胞株存活之探討------------------ 46
2.不同濃度之處理物對B16-F10黑色素細胞株形態影響之探討------------------------------ 47
3.不同濃度之處理物對B16-F10黑色素細胞株抑制黑色素生成之探討------------------------ 47
4.不同濃度之處理物對B16-F10黑色素細胞株抑制酪胺酸?酵素活性之探討------------------ 50
5.不同處理物對B16-F10黑色素細胞株中黑色素生成路徑相關酵素的蛋白質表現差異之探討---- 53
6.不同處理物對B16-F10黑色素細胞株中黑色素生成路徑相關酵素的基因表現差異之探討------ 56
7.不同濃度之處理物對B16-F10黑色素細胞株MITF蛋白位置之探討-------------------------- 57
8.不同處理物對B16-F10黑色素細胞株與XB-2角質細胞Co-culture之黑色素小體轉移之探討---- 59
9.不同處理物對XB-2角質細胞株增生影響之探討----------------------------------------- 60
10.結論------------------------------------------------------------------------------ 60
11.未來研究方向---------------------------------------------------------------------- 61
第六章:參考文獻--------------------------------------------------------------------- 63
表----------------------------------------------------------------------------------- 78
圖----------------------------------------------------------------------------------- 80
自述--------------------------------------------------------------------------------- 107
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