臺灣博碩士論文加值系統

Butein (3,4,2’,4’-tetrahydroxychalcone)紫鉚花素存在於腰果的樹皮、降香黃壇的心材及漆樹等相關植物萃取之漢方中藥材，並且紫鉚花素是一種從 Rhus verniciflua Stokes 抽取的多酚類物質。由本地萃取出來的 Butein主要包含了強力的 chalcone，其好處包括抗氧化、抗發炎，以及內皮依賴性血管舒張的誘發，抑制蛋白質激酶和抑制在多種人類腫瘤細胞的抗癌活性。雖然 Butein 已經被證明會在許多的癌症上誘導癌細胞的凋亡，但 Butein對於黑色素細胞瘤的遷移和侵襲的影響目前是沒有報導的。其目的是研究 Butein 在腫瘤的進展過程中的侵襲和轉移的抑制效果。本研究還證明了 Butein抑制 B16F10 黑色素細胞的移行和侵襲在 trans-well方法和 scratch方法的模式中呈現一個劑量依賴性在(1-10 μM)。並Western blot分析顯示，Butein (1-10 μM)會抑制 B16F10 黑色素細胞的 ERK1/2活化。此外，在體內結果發現，Butein會減少 B16F10 黑色素細胞的的轉移。總之，這是首次研究證明，Butein作為抗癌藥用於治療是透過抑制遷移和侵襲在 B16F10 黑色素細胞。

3,4,2’,4’-Tetrahydroxychalcone (butein) is a type of chalcone derivatives which is a group of flavonoid. Flavonoid is one such agent of polyphenol from the numerous plants tissues including stem-bark of cashews (Semecarpus anacardium), the heartwood of Dalbergia odorifera and including Caragana jubata and Rhus verniciflua stokes in traditional Chinese and Tibetan medicinal herbs. The extract of native butein predominantly contains the potent chalcone with the reported benefits include antioxidant activity, anti-inflammatory activity, elicitation of endothelium-dependent vasodilation, protein kinase inhibition and anti-tumor active inhibition in a variety of human tumor cells. Although Butein has been shown to induce apoptosis in many cancer cells, the effects of butein on the migration and invasion of melanoma are not reported. The objective was to examine the inhibitory effects of Butein on invasion and metastasis during tumor progression.
Our results also demonstrated that butein inhibited B16F10 migration and invasion as determined by two-dimensional migration scratch assay and transwell model in a concentration-dependent manner (1-10 μM). Western blotting analysis indicated that butein (1-10 μM) inhibite the activation of ERK1/2 in B16F10. In addition, in vivo results showed that butein attenuated metastasis of B16F10.
In conclusion, this is the first study to demonstrate that butein as an anticancer agent for the treatment of B16 melanoma cell through inhibiting migration and invasion.

目 錄

中文摘要 2
英文摘要 3
縮寫表 4
目 錄 5
圖索引 7
Chapter1 簡介 8
1.1 癌化過程簡介 8
1.1.1 癌症轉移過程的階段 9
1.1.2 癌細胞轉移抑制劑 10
1.1.3 抗腫瘤藥物機轉 10
1.2 黑色素細胞瘤 11
1.2.1 皮膚的功能 11
1.2.2 疾病原因 11
1.2.3 危險因子 12
1.2.4 惡性黑色素細胞瘤 12
1.2.5 黑色素細胞瘤的分期 13
1.2.6 治療方式 15
1.3 Butein 16
Chapter2 方法跟材料 22
2.1 材料 22
2.2 細胞培養 (Cell Culture) 22
2.2.1 細胞培養 23
2.2.2 繼代培養 24
2.2.3 細胞計數 24
2.3 細胞存活率測定 26
2.4 傷口癒合分析法 26
2.5 細胞轉移能力分析 27
2.6 西方墨點法 27
2.6.1 抽取細胞蛋白質 27
2.6.2 測定蛋白質濃度 28
2.6.3 SDS-聚丙烯醯銨膠體電泳的製作 28
2.6.4 電泳 28
2.6.5 轉漬 29
2.6.6 免疫染色 29
2.6.7 呈色與壓片 30
2.7 動物實驗 30
2.8 統計分析 29
Chapter3 結果 31
3.1 紫鉚花素(Butein)對於黑色素細胞(B16F10)造成之細胞毒性 31
3.2 紫鉚花素(Butein)利用 wound healing 對於黑色素細胞(B16F10)的移行性（Migration） 31
3.3 紫鉚花素(Butein)利用 Boyden chamber 對於黑色素細胞(B16F10)的轉移潛力測試 32
3.4 紫鉚花素(Butein)對於黑色素細胞(B16F10)中(ERK 1/2)蛋白表現增加的影響
33
3.5 紫鉚花素(Butein)對於黑色素細胞(B16F10)的體內動物模式中的轉移能力 34
Chapter4 討論 43
參考文獻 48


圖索引
Fig.1.1 癌症轉移的過程 18
Fig.1.2.1 皮膚分層以及皮膚的主要構造 18 Fig.1.2.2惡性黑色素細胞瘤生長圖 18
Fig.1.2.3 Stage 0 Melanona 19
Fig.1.2.4 Stage I Melanona 19
Fig.1.2.5 Stage II Melanona 19
Fig.1.2.6 Stage III Melanona 20
Fig.1.2.7 Stage IV Melanona 20
Fig.1.4 Butein 21
Fig.3.1 紫鉚花素(Butein)對於黑色素細胞(B16F10) 造成之細胞毒性量化圖35 Fig.3.2 紫鉚花素(Butein)利用 wound healing 對於黑色素細胞(B16F10)的移 行性（Migration） 36
Fig.3.3 紫鉚花素(Butein)利用 wound healing 對於黑色素細胞(B16F10)的移 行性（Migration）量化圖 37
Fig.3.4 紫鉚花素(Butein)利用 Boyden chamber 對於黑色素細胞(B16F10)的 轉移潛力測試 38 Fig.3.5 紫鉚花素(Butein)利用 Boyden chamber 對於黑色素細胞(B16F10)的 轉移潛力測試 量化圖 39
Fig.3.6 紫鉚花素(Butein)對於黑色素細胞(B16F10)中(ERK 1/2)蛋白表現增 加的影響 40
Fig.3.7紫鉚花素(Butein)對於黑色素細胞(B16F10)的體內動物模式中的轉移能力 41
Fig.3.8紫鉚花素(Butein)對於黑色素細胞(B16F10)的體內動物模式中的轉移能力量化圖 42

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