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研究生:彭芷瑜
研究生(外文):Chih-Yu Peng
論文名稱:咖啡酸苯乙酯抑制口腔癌細胞轉移和侵襲研究
論文名稱(外文):Inhibitory effects of Caffeic Acid Phenethyl Ester on Oral Cancer Cell Metastasis and Invasion
指導教授:周明勇周明勇引用關係
指導教授(外文):Ming-yuan Chou
學位類別:博士
校院名稱:中山醫學大學
系所名稱:牙醫學系碩士班
學門:醫藥衛生學門
學類:牙醫學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:88
相關次數:
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咖啡酸苯乙酯 ( caffeic acid phenethyl ester, CAPE ) 分子式為 C17H16O4 ,是蜂膠內主要活性成分之一。目前已經被證實具有抗發炎、抗氧化活性、抗病毒及抑制癌細胞生長等活性。在口腔癌方面,目前僅知 CAPE 在高濃度下會造成口腔癌細胞死亡,而 CAPE 是否可抑制口腔癌細胞株的侵襲與轉移,以及其在口腔癌細胞株內的分子機制,目前仍不清楚。本論文的目的在探討 CAPE 對於口腔鱗狀細胞癌細胞株 SCC-9 侵襲能力的影響與機制。結果顯示, CAPE 在無細胞毒殺濃度下 (0 μM to 40μM) ,會減弱 SCC-9 轉移與侵略性。西方墨點法與明膠酶譜分析更進一步發現, CAPE 會減低基質金屬蛋白酶-2 ( matrix metalloproteinase-2,MMP-2 ) 的表現與其酵素活性。 因為 CAPE 有抑制 MMP-2 表現的能力,所以對其抑制因子tissue inhibitor of metalloproteinase-2 (TIMP-2) 有活化與正調控的效果。同時也會減低focal adhesion kinase ( FAK ) 的磷酸化,以及其下游 p38/MAPK 與 JNK 等的磷酸化。 實驗結果的資料顯示,CAPE可能可以作為預防或治療口腔癌轉移的化學治療製劑。

Caffeic acid phenethyl ester (CAPE), an active component extracted from honeybee hives, exhibits anti-inflammatory and anticancer activities. However, the molecular mechanism by which CAPE affects oral cancer cell metastasis has yet to be elucidated. In this study, we investigated the potential mechanisms underlying the effects of CAPE on the invasive ability of SCC-9 oral cancer cells. Results showed that CAPE attenuated SCC-9 cell migration and invasion at noncytotoxic concentrations (0 μM to 40μM). Western blot and gelatin zymography analysis findings further indicated that CAPE downregulated matrix metalloproteinase-2 (MMP-2) protein expression and inhibited its enzymatic activity. CAPE exerted its inhibitory effects on MMP-2 expression and activity by upregulating tissue inhibitor of metalloproteinase-2 (TIMP-2) and potently decreased migration by reducing focal adhesion kinase (FAK) phosphorylation and the activation of its downstream signaling molecules p38/MAPK and JNK. These data indicate that CAPE could potentially be used as a chemoagent to prevent oral cancer metastasis.


目錄
摘要....................... I
Abstract..................... II
縮寫表......................IV
壹、 緒論.....................1
1. 口腔鱗狀細胞癌( oral squamous cell carcinoma ).. 1
2. 腫瘤轉移 ( metastasis )............3
3. 細胞凋亡 (apoptosis )............5
4. 基質金屬蛋白酶 ( Matrix Metalloproteinases )..9
5. 訊息傳遞路徑 ( Signal Transduction Pathway )..14
6. 咖啡酸苯乙酯( Caffeic acid phenethyl ester )...18
貳、 研究目的...................20
參、 研究內容與方法................22
1. 實驗細胞株 (Cell lines)......... ..22
2. 細胞培養 (cell culture)............22
3. 細胞存活率分析 ( Analysis of cell viability )...25
4. Annexin V / PI 雙染法............26
5. 體外腫瘤轉移試驗 ( Tumor metastasis assay in
vitro )..................27
6. 受質十二烷基硫酸鈉聚丙烯醯胺凝膠電泳(Substrate
SDS-PAGE)................ 30
7. 西方點墨法 (Western blot).......... 31
8. 統計分析 (Statistical analysis ).........33
肆、結果.....................34
1. 咖啡酸苯乙酯對於人類口腔鱗狀細胞癌 SCC-9 細
胞存活率的影響............. 34
2.咖啡酸苯乙酯抑制人類口腔鱗狀細胞癌 SCC-9 移動
能力的分析................ 35
3.咖啡酸苯乙酯抑制人類口腔鱗狀細胞癌 SCC-9 轉
移與侵襲能力的分析............ 35
4.咖啡酸苯乙酯抑制人類口腔鱗狀細胞癌 SCC-9分
泌MMP-2活性的能力分析.........36
5.咖啡酸苯乙酯抑制人類口腔鱗狀細胞癌 SCC-9 內
生性MMP-2與其內生性抑制劑TIMP-2的蛋白表現
    .....................36
6. 咖啡酸苯乙酯對人類口腔鱗狀細胞癌 SCC-9其
Focal Adhesion Kinase ( FAK ) 活性的影響...37
7. 咖啡酸苯乙酯對人類口腔鱗狀細胞癌 SCC-9其
MAPK與Akt路徑的影響..........37
伍、討論.....................39
陸、參考文獻...................45
柒、圖表與圖表說明................55
捌、已發表的論文.................69
圖表目錄
圖一 咖啡酸苯乙酯對於人類口腔鱗狀細胞癌 SCC-9 細胞存活率
的影響.......................55
圖二 咖啡酸苯乙酯對於人類口腔牙齦纖維母細胞存活率的影響.56
圖三 利用流式細胞儀分析證明咖啡酸苯乙酯不會造成人類口腔鱗
狀細胞癌 SCC-9 細胞凋亡..............57
圖四 咖啡酸苯乙酯不會影響人類口腔鱗狀細胞癌 SCC-9 細胞內
凋亡蛋白酶的活性..................58
圖五 咖啡酸苯乙酯對於人類口腔鱗狀細胞癌SCC-9 細胞移動能力
的影響.......................59
圖六 咖啡酸苯乙酯對於人類口腔鱗狀細胞癌 SCC-9 細胞轉移能
力的影響......................60
圖七 咖啡酸苯乙酯對於人類口腔鱗狀細胞癌 SCC-9 細胞侵襲能
力的影響......................61
圖八 咖啡酸苯乙酯對於人類口腔鱗狀細胞癌 SCC-9 細胞 MMP-2
酵素活性之影響...................62
圖九 咖啡酸苯乙酯對於人類口腔鱗狀細胞癌 SCC-9 細胞分泌
MMP-2與內生性抑制劑TIMP-2的蛋白表現之影響....63
圖十 咖啡酸苯乙酯對於人類口腔鱗狀細胞癌 SCC-9 細胞其訊息
傳遞蛋白FAK磷酸化表現之影響...........64
圖十一 咖啡酸苯乙酯對於人類口腔鱗狀細胞癌 SCC-9 細胞其訊息
傳遞蛋白ERK磷酸化表現之影響...........65
圖十二 咖啡酸苯乙酯對於人類口腔鱗狀細胞癌 SCC-9 細胞其訊息
傳遞蛋白p38磷酸化表現之影響...........66
圖十三 咖啡酸苯乙酯對於人類口腔鱗狀細胞癌 SCC-9 細胞其訊息
傳遞蛋白JNK磷酸化表現之影響...........67
圖十四 咖啡酸苯乙酯對於人類口腔鱗狀細胞癌 SCC-9 細胞其訊息
傳遞蛋白Akt磷酸化表現之影響........... 68


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