臺灣博碩士論文加值系統

皮膚和黑色素分佈及合成黑色素的訊息調控間關係密切，黑色素細胞是合成黑色素的重要調控者，因此，研究黑色素瘤細胞機制及其應用對於後天性的色素沉著或缺失的改善有其重要性。本研究中，我們探討柑橘屬植物中的黃烷酮衍生物及 PPARα 活化劑fenofibrate 在鼠類B16-F10黑色素瘤細胞之黑色素生成作用機制。
柑橘屬所含之黃烷酮衍生物主要有naringenin、naringin、narirutin、hesperetin、hesperidin及neohesperidin。研究結果顯示，黃烷酮的醣苷元化合物 naringenin 及 hesperetin 具有明顯促進B16-F10細胞生成黑色素之作用，其中以 naringenin 之效果最明顯。Naringenin 濃度增加，其細胞內的酪胺酸酶活性及MITF的表現量也增加。此外，naringenin 會增加細胞內 cAMP 的含量， naringenin 所誘導的黑色素生成可完全地被 (PKA 的抑制劑) H89 所阻斷，而 (MEK 的抑制劑) U0126、(phosphatidylinositol 3-kinase的抑制劑) LY294002會促進 naringenin 所誘導的黑色素生成作用，以上的結果顯示，naringenin 促進黑色素生成的作用可能經由 cAMP 的機制及 MAPK 或 PI3K 路徑調控而來影響黑色素的合成。
PPAR 是細胞核賀爾蒙接受器的家族之ㄧ，其活化劑在皮膚表皮的調控平衡上扮演著重要的角色。研究結果顯示，只有 PPARα 的活化劑 (fenofibrate) 會明顯地抑制黑色素生成，而 PPARβ 的活化劑 (GW0742) 及 PPARγ 的活化劑 (ciglitazone) 則沒有此現象。此外，Fenofibrate 會抑制 MITF 的表現但確不會影響細胞內酪胺酸酶的活性，因此本研究數據顯示 PPARα 的活化劑可能藉由下降調節 MITF 的表現來減少 B16-F10 之黑色素生成。

The color of mammalian skin and hair is determined by a number of factors, the most obvious phenotypical characteristics of which is the distribution of melanin pigment. Therefore, melanocytes act as important regulators for skin pigmentation and targets to study the mechanism of melanoma. In the present study, we investigated the action mechanisms of Citrus flavonoids and peroxisome proliferator-activated receptors (PPARs) activators on melanogenesis in B16-F10 melanoma cells.
The most abundant Citrus flavonoids are flavanones, such as naringenin, naringin, narirutin, hesperetin, hesperidin and neohesperidin. We showed that the synthesis of melanin was strongly increased by naringrnin or hesperetin, the flavanone aglycone. In this study, the cellular tyrosinase activity also enhanced by naringenin in a concentration-dependent manner. Moreover, naringenin increased the intracellular cAMP level and the naringenin-induced melanogenesis was completely abolished by H89, an inhibitor of protein kinase A. Furthermore, we determined the roles of mitogen-activated protein kinases (MAPKs) on naringenin-induced melanogenesis. The melanin contents of naringenin-treated cells were significantly raised by the addition of U0126 and LY294002, an inhibitor of MAPK kinase and phosphatidylinositol 3-kinase (PI3K), respectively. These results suggest that naringenin increases melanin synthesis through a cAMP-dependent mechanism and probably via the modulation of MAPK or PI3K pathway.
PPARs (peroxisome proliferator-activated receptors) are members of the nuclear hormone receptor superfamily. Agonists of PPARs have been known to play an important role in epidermis homeostasis. However, the molecular events of PPARs have not been well investigated in melanocytes. We evaluated the effects of PPAR agonists on the melanogenic activities. The data showed that only the PPARα activator (fenofibrate) significantly inhibited the melanin synthesis, not PPARβ activator (GW0742) or PPARγ activator (ciglitazone). In addition, fenofibrate also diminished the expression of MITF but did not alter the cellular tyrosinase activity. The present study implies that PPARα activator reduces melanin synthesis via the down-regulation of MITF in B16-F10 melanoma cells. PPARα may contribute to the regulation of melanogenesis.

目錄
縮寫表
中文摘要………………………………………………………………..Ⅰ
英文摘要……………………………………………………………..…Ⅱ
圖目錄……………………………………………………………..……Ⅵ
第一章、 緒論………………………………………………………...8
一、 黑色素細胞與黑色素瘤………………………………………..8
二、 黑色素生成作用………………………………………………..9
三、 黃烷酮衍生物…………………………………………………11
四、 PPAR…………………………………………………………..13
五、 研究動機與目的………………………………………………15
第二章、 材料與方法……………………………………………….16
一、 實驗材料………………………………………………………16
(一) 材料……………………………………………………………..16
(二) 溶液……………………………………………………………..17
二、 實驗方法………………………………………………………18
(一) 細胞培養及細胞存活率分析……………………………………...18
(二) 黑色素含量測定…………………………………………………...18
(三) 酪胺酸酶活性測定……………………………………………...…18
A. Cell-free 酪胺酸酶活性測定…………………………………18
B. 細胞內酪胺酸酶活性測定……………………………………18
C. DOPA staining 測定…………………………………………..18
(四) 西方點墨法…………..…………………………………………….19
(五) cAMP 濃度測定…..…..…………………………………………...20
(六) 統計方法…………………………………………………………...20
第三章、 結果………………………………………………………..21
第一節、黃烷酮衍生物對鼠類 B16-F10 黑色素瘤細胞之黑色素生成作用探討………………………………..………………………..……..21
一、黃烷酮衍生物對 B16-F10 細胞存活率之影響…………………21
二、黃烷酮衍生物對 B16-F10 細胞黑色素生成效果之影響………21
三、Naringenin 對 B16-F10 細胞酪胺酸酶之影響…………………22
四、Naringenin 促進 B16-F10 細胞黑色素生成之路徑探討………22
第二節、Fenofibrate 對鼠類黑色素瘤 B16-F10 細胞之黑色素生成作用探討…………………………………………………………………..24
一、PPAR 活化劑對 B16-F10 細胞存活率之影響…………………24
二、PPAR 活化劑對 B16-F10 細胞黑色素生成效果之影響………24
三、PPAR 活化劑對 B16-F10 細胞酪胺酸酶之影響…………..…..24
四、Fenofibrate 抑制B16-F10細胞產生黑色素之路徑探討………..25
第四章、 討論………………………………………………………..26
第五章、 參考文獻………………………..…………………………29
圖目錄
圖一、黑色素結構……………………………………………………..38
圖二、黑色素生成作用………………………………………………..39
圖三、PKA 路徑與黑色素生成作用之調控機制模式圖……………40
圖四、cAMP 造成黑色素生成的訊號傳遞模式圖…………………..41
圖五、MITF 與黑色素生成作用之調控機制…………………….…..42
圖六、類黃酮之分類結構式…………………………….……………..43
圖七、柑橘類黃烷酮非醣苷及醣苷的結構式…………………………44
圖8 Naringenin、narirutin及naringin 對B16-F10 細胞存活率之影響.…………………………...……..……………………...…………….45
圖9 Hesperetin、hesperidin 及 neohesperidin 對B16-F10 細胞存活率之影響.…………………………………...………..……………………46
圖 10 Naringenin 對B16-F10 細胞增生之影響………….………….47
圖 11 Naringenin、narirutin、naringin、hesperetin、hesperidin及 neohesperidin 對B16-F10 細胞黑色素生成效果之影響…………....48
圖 12 Naringenin不同時間作用下的 B16-F10 細胞黑色素生成效果之影響…………………………………………………………………..49
圖 13 Naringenin不同濃度作用下的 B16-F10 細胞黑色素生成效果之影響……………………………………………………………….….50
圖 14 Naringenin對B16-F10 細胞酪胺酸酶直接效果之影響…..….51
圖 15 Naringenin對B16-F10 細胞內酪胺酸酶之影響………………52
圖 16 Naringenin對B16-F10 細胞中 MITF 的表現量之影響….….53
圖 17 Naringenin對B16-F10 細胞中 cAMP 的含量之影響……….54
圖 18 Naringenin對B16-F10 細胞中 pAKT 的表現量之影響…….55
圖 19 Naringenin及LY294002對B16-F10 細胞黑色素生成效果之影響………………………….……………………………………….……56
圖 20 Naringenin及LY294002對B16-F10 細胞內酪胺酸酶之影響.57
圖 21 Naringenin及H89對B16-F10 細胞黑色素生成效果之影響……………………………………...………………………………...58
圖 22 Naringenin及H89對B16-F10 細胞內酪胺酸酶之影響………59
圖 23 Naringenin及U0126對B16-F10 細胞黑色素生成效果之影響………………………………………………………………………..60
圖 24 PPAR 活化劑對 B16-F10 細胞存活率之影響…..…………...61
圖 25 PPAR 活化劑對B16-F10 細胞黑色素生成效果之影響..…....62
圖 26 Fenofibrate 對B16-F10細胞酪胺酸酶直接效果之影響………63
圖 27 Fenofibrate對B16-F10 細胞內酪胺酸酶之影響……...………64
圖 28 Fenofibrate對B16-F10 細胞中 MITF 的表現量之影響……..65
圖 29 PPAR 活化劑及抑制劑對B16-F10 細胞黑色素生成效果之影響………………………………………………………………………..66

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