臺灣博碩士論文加值系統

已知TRP1和TRP2是黑色素細胞中eumelanin生成過程之重要酵素，且二者之表現受上游基因Mitf的活化而向上調控。又已知甲型硫辛酸會藉由調控細胞中麩胱甘肽含量，使B16-F1黑色素瘤細胞內外之eumelanin生合成降低，並減少細胞內、外黑色素含量，因此，本實驗主要探討(1)不同濃度甲型硫辛酸處理B16-F1黑色素瘤細胞72小時後，對細胞內、外黑色素生合成之影響。(2)以西方墨點分析法檢測不同濃度甲型硫辛酸處理B16-F1黑色素瘤細胞72小時後，對細胞中eumelanin生合成相關基因，包括TRP1、TRP2、Mitf表現之影響。實驗結果發現：(1)隨著甲型硫辛酸濃度增加，會減少B16-F1黑色素瘤細胞內、外黑色素含量。(2) 400μM甲型硫辛酸之處理會降低B16-F1黑色素瘤細胞中TRP2及Mitf之表現量，然而對TRP1之表現則無明顯影響。顯示甲型硫辛酸可能藉由抑制Mitf的表現，進而抑制下游基因TRP2之表現，使 B16-F1黑色素瘤細胞內、外黑色素含量降低。

It has been confirmed that TRP1 and TRP2 are the important enzymes involved in eumelanogenesis of melanocytes, and the expressions of TRP1 and TRP2 genes are up-regulated by activation of upstream gene, Mitf. Alpha-Lipoic acid decreased eumelanogenesis by regulating the glutathione content in cells, and reduced the intra/ extracellular melanin content of B16-F1 melanoma cells. Therefore, our study was to evaluate (1) the intra/extracellular melanin biosynthesis of B16-F1 melanoma cells when treated with different concentrations of Alpha-lipoic acid at 72 hours, (2) the expressions of eumelanogenesis related genes, including TRP1, TRP2 and Mitf of B16-F1 melanoma cells when treated with different concentrations of Alpha-lipoic acid at 72 hours. Our results has shown that (1) the intra/extracellular melanin content were decreased by increasing the concentrations of Alpha-lipoic acid. (2) 400μM Alpha-lipoic acid also reduced the expressions of TRP2 and Mitf of B16-F1 melanoma cells, but TRP1 expression remained unchanged. The results suggested that Alpha-lipoic acid inhibited the expression of Mitf, and further inhibited the expression of a downstream gene, TRP2, to decrease intra/ extracellular melanin content of B16-F1 melanoma cells.

目 錄
目錄----------------------------------------------------------I
表目錄------------------------------------------------------VII
圖目錄-------------------------------------------------------IV
中文摘要----------------------------------------------------XII
英文摘要---------------------------------------------------XIII
壹、緒論------------------------------------------------------1
一、黑色素生合成步驟------------------------------------------1
二、Eumelanin 生合成相關基因與色素形成之關係------------------5
三、Eumelanin生合成相關之基因表現受α-melanocyte-
stimulating hormone(α-MSH) 及agouti signal protein(ASP)所調控-8
(一)、α-MSH之調控---------------------------------------------8
(二)、ASP之調控-----------------------------------------------9
四、甲型硫辛酸對黑色素生合成及Eumelanin生合成相關基因表現之影響-----------------------------------------------------------12
貳、研究目的-------------------------------------------------14
參、實驗設計與流程-------------------------------------------15
一、實驗設計-------------------------------------------------15
二、實驗流程-------------------------------------------------16
肆、實驗方法-------------------------------------------------17
一、細胞株及細胞培養-----------------------------------------17
二、細胞內、外黑色素量之觀察---------------------------------18
三、細胞計數-------------------------------------------------19
四、細胞內蛋白值之萃取---------------------------------------19
五、蛋白質定量分析-------------------------------------------20
六、SDS-膠體電泳及西方墨點分析-------------------------------21
七、西方墨點分析結果之量化-----------------------------------22
八、分析統計-------------------------------------------------23
伍、實驗結果-------------------------------------------------24
一、繼代培養代數及甲型硫辛酸對B16-F1黑色素瘤細胞生長之影響---24
(一)、繼代培養代數對細胞生長之影響---------------------------24
(二)、甲型硫辛酸對細胞生長之影響-----------------------------25
二、繼代培養代數及甲型硫辛酸對B16-F1黑色素瘤細胞形態之影響---33
(一)、繼代培養代數對細胞形態之影響---------------------------33
(二)、甲型硫辛酸對細胞形態之影響-----------------------------34
三、繼代培養代數及甲型硫辛酸對B16-F1黑色素瘤細胞內黑色素生合成之影響-------------------------------------------------------38
(一)、繼代培養代數對細胞內黑色素含量之影響-------------------38
(二)、甲型硫辛酸對細胞內黑色素含量之影響---------------------39
四、繼代培養代數及甲型硫辛酸對B16-F1黑色素瘤細胞外黑色素生合成之影響-------------------------------------------------------41
(一)、繼代培養代數對細胞外黑色素含量之影響-------------------41
(二)、甲型硫辛酸對細胞外黑色素含量之影響---------------------50
五、B16-F1黑色素瘤細胞內外黑色素含量之關係-------------------56
(一)、繼代培養代數增加時，細胞內外黑色素含量之關係-----------56
(二)、甲型硫辛酸濃度增加時，細胞內外黑色素含量之關係---------56
六、B16-F1黑色素瘤細胞外黑色素含量與細胞生長之關係-----------58
(一)、繼代培養代數增加時，細胞外黑色素含量與細胞生長之關係---58
(二)、甲型硫辛酸濃度增加時，細胞外黑色素含量與細胞生長之關係-59
七、繼代培養代數及甲型硫辛酸對B16-F1黑色素瘤細胞中eumelanin生合成相關基因表現之影響----------------------------------------66
(一)、繼代培養代數對細胞中eumelanin生合成相關基因表現之影響-66
(二)、甲型硫辛酸對細胞中eumelanin生合成相關基因表現之影響---69
八、B16-F1黑色素瘤細胞外黑色素含量與細胞中eumelanin生合
成相關基因表現之關係----------------------------------------87
(一)、繼代培養代數增加時，細胞外黑色素含量與細胞中eumelanin 生合成相關基因表現之關係--------------------------------------87
(二)、甲型硫辛酸濃度增加時，細胞外黑色素含量與細胞中eumelanin 生合成相關基因表現之關係------------------------------------88
陸、討論-----------------------------------------------------93
一、繼代培養代數及甲型硫辛酸對B16-F1黑色素瘤細胞生長之影響---93
(一)、繼代培養代數對細胞生長之影響---------------------------93
(二)、甲型硫辛酸對細胞生長之影響-----------------------------94
二、繼代培養代數及甲型硫辛酸對B16-F1黑色素瘤細胞內、外黑色素生合成與細胞形態之影響-----------------------------------------95
(一)、細胞內黑色素生合成-------------------------------------95
(1)、繼代培養代數對細胞內黑色素生合成之影響------------------95
(2)、甲型硫辛酸對細胞內黑色素生合成之影響-------------------96
(二)、細胞外黑色素生合成------------------------------------97
(1)、繼代培養代數對細胞外黑色素生合成之影響-----------------97
(2)、甲型硫辛酸對細胞外黑色素生合成之影響-------------------98
(三)、細胞形態----------------------------------------------99
(1)、繼代培養代數對細胞形態之影響---------------------------99
(2)、甲型硫辛酸對細胞形態之影響----------------------------100
三、甲型硫辛酸抑制黑色素生合成之作用較細胞老化所降低之黑色素生合成作用明顯----------------------------------------------101
四、繼代培養代數及甲型硫辛酸對B16-F1黑色素瘤細胞中eumelanin生合成相關基因表現之影響--------------------------------------102
(一)、繼代培養代數對細胞中eumelanin生合成相關基因表現之影響-102
(二)、甲型硫辛酸對細胞中eumelanin生合成相關基因表現之影響
之影響------------------------------------------------------103
柒、結論---------------------------------------------------106
捌、參考文獻-----------------------------------------------107
玖、附錄---------------------------------------------------118
一、實驗材料-----------------------------------------------118
二、實驗常用溶液之配製-------------------------------------122
三、冷光螢光影像分析系統量化方式---------------------------127
四、冷光螢光影像分析系統量化結果---------------------------128
表 目 錄
表一、Human melanoma cell line 中tyrosinase 相關基因表現、dopa oxidase 活性及黑色素含量之比較-------------------------------7
表二、繼代培養代數對B16-F1黑色素瘤細胞生長之影響-------------26
表三、繼代培養代數與細胞生長之相關性-------------------------28
表四、甲型硫辛酸對各代數B16-F1黑色素瘤細胞生長之影響---------29
表五、0∼200μM甲型硫辛酸劑量與細胞生長之相關性---------------31
表六、繼代培養代數對B16-F1黑色素瘤細胞外黑色素含量之影響-----47
表七、繼代培養代數與細胞外黑色素含量之相關性-----------------49
表八、甲型硫辛酸對各代數B16-F1 黑色素瘤細胞外黑色素含量之影響---------------------------------------------------------------52
表九、甲型硫辛酸劑量與細胞外黑色素含量之相關性---------------54
表十、繼代培養代數增加時，細胞外黑色素含量與細胞生長之相關性-61
表十一、甲型硫辛酸劑量增加時，細胞外黑色素含量與細胞生長之相關性-----------------------------------------------------------64
表十二、甲型硫辛酸對各代數B16-F1黑色素瘤細胞中eumelanin生合成相關基因表現之影響(目測量化)-----------------------------------72
表十三、繼代培養代數與細胞中eumelanin生合成相關基因表現之相關性-----------------------------------------------------------74
表十四、繼代培養代數增加時，細胞中TRP2-U與TRP2-L表現之相關性-76
表十五、繼代培養代數增加時，細胞中Mitf-U與Mitf-L表現之相關性
-------------------------------------------------------------78
表十六、甲型硫辛酸濃度與細胞中eumelanin生合成相關基因表現之相關性-----------------------------------------------------------81
表十七、甲型硫辛酸濃度增加時，細胞中TRP2-U與TRP2-L表現之相關性--------------------------------------------------------------83
表十八、甲型硫辛酸濃度增加時，細胞中Mitf-U與Mitf-L表現之相關性--------------------------------------------------------------85
表十九、繼代培養代數增加時，細胞外黑色素含量與細胞中eumelanin生合成相關基因表現之相關性-------------------------------------89
表二十、甲型硫辛酸濃度增加時，細胞外黑色素含量與細胞中eumelanin生合成相關基因表現之相關性-----------------------------------91
表二十一、甲型硫辛酸對各代數B16-F1黑色素瘤細胞中eumelanin生合成相關基因表現之影響(冷光螢光影像分析系統量化結果)------------130
圖 目 錄
圖一、黑色素之運送--------------------------------------------3
圖二、黑色素生合成途徑----------------------------------------4
圖三、α-MSH及ASP對melanogenic gene之調控---------------------11
圖四、繼代培養代數對B16-F1黑色素瘤細胞生長之影響(表二量化圖)-27
圖五、繼代培養代數與細胞生長之相關性-------------------------28
圖六、甲型硫辛酸對各代數B16-F1黑色素瘤細胞生長之影響
(表四量化圖)-------------------------------------------------30
圖七、0∼200μM甲型硫辛酸劑量與細胞生長之相關性---------------32
圖八、甲型硫辛酸對年輕代數B16-F1黑色素瘤細胞形態之影響-------35
圖九、甲型硫辛酸對中年代數B16-F1黑色素瘤細胞形態之影響-------36
圖十、甲型硫辛酸對老年代數B16-F1黑色素瘤細胞形態之影響-------37
圖十一、甲型硫辛酸對各代數B16-F1黑色素瘤細胞內黑色素含量之
影響---------------------------------------------------------40
圖十二、甲型硫辛酸處理24小時對各代數B16-F1黑色素瘤細胞培養液顏色之影響-----------------------------------------------------44
圖十三、甲型硫辛酸處理48小時對各代數B16-F1黑色素瘤細胞培養液顏色之影響-----------------------------------------------------45
圖十四、甲型硫辛酸處理72小時對各代數B16-F1黑色素瘤細胞培養液顏色之影響-----------------------------------------------------46
圖十五、繼代培養代數對B16-F1黑色素瘤細胞外黑色素含量之影響(表六量化圖)------------------------------------------------------48
圖十六、繼代培養代數與細胞外黑色素含量之相關性---------------49
圖十七、甲型硫辛酸對各代數B16-F1黑色素瘤細胞外黑色素含量之影響(表八量化圖)-------------------------------------------------53
圖十八、甲型硫辛酸劑量與細胞外黑色素含量之相關性-------------55
圖十九、甲型硫辛酸處理72小時對各代數B16-F1黑色素瘤細胞內外黑色素含量之影響(圖十一與圖十四合併比較)-------------------------57
圖二十、繼代培養代數對B16-F1黑色素瘤細胞外黑色素含量及細胞生長之影響(圖十五與圖七合併比較)---------------------------------60
圖二十一、繼代培養代數增加時，細胞外黑色素含量與細胞生長之相關性-----------------------------------------------------------62
圖二十二、甲型硫辛酸對B16-F1黑色素瘤細胞外黑色素含量及細胞生長之影響(圖十七與圖九合併比較)---------------------------------63
圖二十三、甲型硫辛酸劑量增加時，細胞外黑色素含量與細胞生長之相關性---------------------------------------------------------65
圖二十四、甲型硫辛酸處理後，各代數B16-F1黑色素瘤細胞中eumelanin生合成相關基因之表現-----------------------------------------71
圖二十五、繼代培養代數對B16-F1黑色素瘤細胞中eumelanin生合成相關基因表現之影響(表十二量化圖)---------------------------------73
圖二十六、繼代培養代數與細胞中eumelanin生合成相關基因表現之相關性-----------------------------------------------------------75
圖二十七、繼代培養代數增加時，細胞中TRP2-U與TRP2-L表現之相關性--------------------------------------------------------------77
圖二十八、繼代培養代數增加時，細胞中Mitf-U與Mitf-L表現之相關性--------------------------------------------------------------79
圖二十九、甲型硫辛酸對各代數B16-F1黑色素瘤細胞中eumelanin生合成相關基因表現之影響(表十二量化圖)-----------------------------80
圖三十、甲型硫辛酸濃度與細胞中eumelanin生合成相關基因表現之相關性-----------------------------------------------------------82
圖三十一、甲型硫辛酸濃度增加時，細胞中TRP2-U與TRP2-L表現之相關性-----------------------------------------------------------84
圖三十二、甲型硫辛酸濃度增加時，細胞中Mitf-U與Mitf-L表現之相關性-----------------------------------------------------------86
圖三十三、繼代培養代數增加時，細胞外黑色素含量與細胞中eumelanin生合成相關基因表現之相關性-----------------------------------90
圖三十四、甲型硫辛酸濃度增加時，細胞外黑色素含量與細胞中eumelanin生合成相關基因表現之相關性--------------------------92
圖三十五、繼代培養代數對B16-F1黑色素瘤細胞中eumelanin生合成相關基因表現之影響(冷光螢光影像分析系統量化結果)(表二十一量化圖)---------------------------------------------------------------131
圖三十六、甲型硫辛酸對各代數B16-F1黑色素瘤細胞中eumelanin生合成相關基因表現之影響(冷光螢光影像分析系統量化結果)(表二十一量化圖)---------------------------------------------------------132

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