臺灣博碩士論文加值系統

魚針草(Anisomeles indica (L.) O. Ktze)主要分布於亞熱帶地區，在台灣主要生長於平地至低海拔山區林緣或荒廢地。民間常用其治療風濕性關節炎、胃腸炎及其它炎症等；而對於魚針草抑制癌細胞生長與轉移(metastasis)，包括移行(migration)與入侵(invasion)作用，至今仍很少被探討。根據衛生署統計資料顯示，2009 年口腔癌的發生率與死亡率為國人十大癌症的第6 位。臨床已證實，口腔癌腫瘤生長速率快，且容易轉移至肺部、淋巴等部位。本研究主要在探討魚針草萃取物 (簡稱AIE)對抑制人類咽部癌FaDu 細胞之生長、移行與入侵作用及其相關機轉進行深入探討。
由癌細胞MTT assay結果顯示，AIE對FaDu細胞有極佳的毒殺效果(24 小時投藥處理的IC50 = 60 μg/ml)。再者，AIE對FaDu細胞之毒殺作用會隨劑量增加而愈趨明顯。由Flow cytometry分析結果顯示，AIE能使FaDu細胞停滯於G2/M生長期。由Western blotting之分析證實，AIE 會經由粒線體路徑向下調控抗凋亡蛋白Bcl-2和Bcl-xL的表現；相對地，亦會向上調控凋亡蛋白Bax、Bad的表現，使其活化下游caspase-3活性，進而誘導FaDu細胞之凋亡。在抑制FaDu細胞移行與入侵方面，由Wound healing assay、Cell migration assay及Cell invasion assay之結果皆顯示，AIE 具有顯著的抑制效果，且隨著投藥劑量的增加，此一抑制效果更為明顯。由Western blotting之分析顯示，p-P38、p-JNK、p-NF-κB、MMP-2、MMP-9及細胞核內的NF-κB表現量皆會隨劑量增加而有降低的趨勢。由於MMP-2及MMP-9為癌細胞移行與入侵作用的重要指標，由ELISA kit assay及gelatin zymography之進一步分析結果顯示，MMP-2及MMP-9的表現量及活性都隨劑量增加而顯著下降。綜合以上結果證明，AIE 確實能有效抑制FaDu 細胞的生長與轉移。

Anisomeles indica (L.) O. Ktze grows mainly in subtropical regions. In Taiwan,it frequently appears in forest edge or abandoned ground of the low-elevationmountains. People often used it as folk medicine to treat rheumatoid arthritis,gastroenteritis, and other inflammatory diseases. However, very few studies reported on the inhibitory effects of A. indica on the proliferation, metastasis, migration and invasion of oral cancer cells. The statistic data from Department of Health show that the incidence and mortality rate of compatriots’ oral cancer patients ranked at the six of the compatriots top ten cancer diseases in Taiwan at 2009. The tumors of oral cancer have been proven clinically to be fast-growth, and are easier to metastasize to lung, lymph nodes and other parts. This study investigates the inhibitory effects of A. indica extract (AIE) on the growth, migration and invasion of FaDu human pharyngeal carcinoma cells and their related mechanisms.
The results of MTT assay for AIE on FaDu cells showed good cytotoxicity on FaDu cells (IC50 = 60 μg/ml at 24-hr treatment) in a dose-dependent manner. By the Flow cytometric analysis, AIE arrested FaDu cells at G2/M phase. Western blotting analysis showed that AIE induced the apoptosis of FaDu cells by down-regulating the anti-apoptotic Bcl-2 and Bcl-xL protein level and up-regulating the apoptotic proteins level of Bax, Bad and the activation of downstream procaspase-3. The results of Wound healing assay, Cell migration assay and Cell invasion assay demonstrated that AIE had significant inhibitory effects on the cell migration and invasion of FaDu cells in a dose-dependent manner. Western blotting analysis showed that the levels of p-P38, p-JNK, p-NF-κB, MMP-2, MMP-9 proteins and the NF-κB in nucleus decreased when the AIE dose increased. Moreover, the results of ELISA kit assay and gelatin zymography on the expression of MMP-2 and MMP-9 showed that both of the MMP-2 and MMP-9 are significantly decline when the dose of AIE increased. In conclusion, the above results illustrated that AIE can inhibit the growth, migration and invasion of the FaDu cells.

中文摘要I
英文摘要II
致謝III
目錄IV
圖目錄VIII
表目錄XI
1. 緒論1
1.1 口腔癌(Oral cancer)1
1.2 天然藥物(Natural medicines)4
1.3 魚針草6
1.4 癌細胞移行(migration)與入侵(invasion)作用9
1.5 研究目的11
2. 研究架構12
3. 材料與方法13
3.1 材料與萃取物之製備13
3.1.1 材料13
3.1.2 萃取物之製備13
3.2 細胞培養條件及方法14
3.2.1 試劑14
3.2.2 藥品配製14
3.2.3 培養條件14
3.2.4 細胞之解凍15
3.2.5 繼代培養15
3.2.6 細胞冷凍保存15
3.2.7 細胞數目計數15
3.3 細胞存活率之測定－MTT assay 16
3.3.1 試劑16
3.3.2 實驗原理16
3.3.3 實驗步驟16
3.4 細胞型態變化觀察16
3.5 西方墨點法17
3.5.1 試劑17
3.5.2 藥品配製19
3.5.3 實驗原理20
3.5.4 細胞溶解液(cell lysates)之製備20
3.5.5 細胞核蛋白萃取21
3.5.6 蛋白質定量21
3.5.7 電泳21
3.5.8 蛋白質電泳semi-dry轉漬(transfer)法21
3.5.9 封阻(Blocking)22
3.5.10 抗體(Antibody)22
3.5.11 免疫染色法 22
3.6 DNA Fragmentation Assay 23
3.6.1 試劑23
3.6.2 藥品配製23
3.6.3 實驗原理23
3.6.3 實驗步驟23
3.7 Trypan Blue Exclusion Assay 24
3.7.1 試劑24
3.7.2 實驗原理24
3.7.3 實驗步驟24
3.8 細胞週期之研究24
3.8.1 試劑24
3.8.2 實驗原理24
3.8.3 實驗步驟25
3.9 細胞凋亡之研究25
3.9.1 試劑25
3.9.2 藥品配製26
3.9.3 實驗原理26
3.9.4 實驗步驟26
3.10 細胞移行能力測定 - Wound healing assay 26
3.10.1 試劑26
3.10.2 實驗原理26
3.10.3 實驗步驟27
3.11 細胞移行能力試驗 - Cell migration assay 27
3.11.1 試劑27
3.11.2 藥品配製27
3.11.3 實驗原理28
3.11.4 實驗步驟28
3.12 細胞入侵能力試驗 - Cell invasion assay 29
3.12.1 試劑29
3.12.2 藥品配製29
3.12.3 實驗原理29
3.12.4 實驗步驟30
3.13 MMP-2 & MMP-9 ELISA Kit Assay 30
3.13.1 試劑30
3.13.2 實驗原理31
3.13.3 實驗步驟31
3.14 MMP-2 & MMP-9 Gelatin Zymography Assay 31
3.14.1 試劑31
3.14.2 藥品配置31
3.14.3 實驗原理33
3.14.4 實驗步驟33
3.15 數據分析33
4. 實驗結果34
4.1 魚針草之萃取分層34
4.2 魚針草萃取物對癌細胞的細胞毒殺作用34
4.2.1 細胞存活檢測(MTT cell viability assay)34
4.2.2 魚針草萃取物對FaDu癌細胞之凋亡影響37
4.2.3 魚針草萃取物對FaDu細胞之蛋白質表現變化44
4.3 抑制口腔癌細胞移行與入侵作用46
4.3.1 魚針草萃取物對FaDu 移行作用之抑制效果46
4.3.2 魚針草萃取物對FaDu 入侵作用之抑制效果51
4.3.3 移行與入侵蛋白之表現53
4.3.4 MMP-2 & MMP-9 ELISA kit定量試驗56
4.3.5 明膠酶譜法(Gelatin zymography assay)57
5. 討論59
6. 結論66
7. 參考文獻67
圖目錄
圖1. 魚針草及花之圖示7
圖2. 不同濃度魚針草萃取物對FaDu口腔癌細胞分別處理(a) 24小時或(b)48小時的型態影響。36
圖3. 不同濃度魚針草萃取物對FaDu口腔癌細胞分別處理48小時或72小時的生長週期分佈之影響38
圖4. 魚針草萃取物於不同濃度處理24 hr 對FaDu癌細胞凋亡程度。(A) Control,(B) Vehicle, (C) 5-Fu (100 μg/ml), (D) 45 μg/ml, (E) 60 μg/ml, (F) 75 μg/ml,(G) 90 μg/ml。LL:左下角象限，UL:左上角象限，LR:右下角象限，UR:右上角象限。未加藥處理的0 μg/ml為Control組，DMSO為Vehicle (濃度為0.50%)。39
圖5. 魚針草萃取物於48 hr不同處理濃度對FaDu癌細胞凋亡程度。(A) Control, (B)Vehicle, (C) 5-Fu (100 μg/ml), (D) 45 μg/ml, (E) 60 μg/ml, (F) 75 μg/ml, (G) 90 μg/ml。LL:左下角象限，UL:左上角象限，LR:右下角象限，UR:右上角象限。未加藥處理的0 μg/ml為Control組，DMSO為Vehicle (濃度0.50%)。40
圖6. 魚針草萃取物於不同濃度處理72 hr對FaDu癌細胞凋亡程度。(A) Control, (B)Vehicle, (C) 5-Fu (100 μg/ml), (D) 45 μg/ml, (E) 60 μg/ml, (F) 75 μg/ml, (G) 90 μg/ml。LL:左下角象限，UL:左上角象限，LR:右下角象限，UR:右上角象限。未加藥處理的0 μg/ml為Control組，DMSO為Vehicle (濃度為0.50%)。41
圖7. 不同濃度的魚針草萃取物(45、60、75、90 μg/ml)，對FaDu口腔癌細胞分別處理48小時或72小時的凋亡程度。LL:左下角象限，UL: 左上角象限， LR:右下角象限，UR:右上角象限。未加藥處理的0 μg/ml為Control組，DMSO為Vehicle (濃度為0.50%)。42
圖8. 不同濃度的魚針草萃取物對FaDu口腔癌細胞處理24 hr後細胞的壞死程度。未加藥處理的0 μg/ml為Control組，DMSO為Vehicle(濃度為0.50%。42
圖9. 以不同濃度魚針草萃取物對FaDu細胞分別處理 (A) 48 hr，及(B) 72 hr的DNA 斷裂情形；其中5-FU為正控制組，濃度為100 μg/ml。43
圖10. 以不同劑量魚針草萃取物對FaDu細胞處理24小時，觀察total 細胞內訊息因子的表現量，5-FU為Positive control。使用β-actin作為loading control。45
圖11. 不同劑量魚針草萃取物及不同處理時間對FaDu癌細胞移行之抑制效應，並以DMSO (劑量0.50%)為Vehicle。以倒立式相位差顯微鏡4倍觀察結果。47
圖12. 不同劑量魚針草萃取物及不同處理時間對FaDu癌細胞移行之抑制效應，並以DMSO (劑量0.50%)為Vehicle。以倒立式相位差顯微鏡4倍觀察結果。48
圖13. 以不同劑量魚針草萃取物處理24 hr對抑制FaDu細胞移行之效應。以倒立式相位差顯微鏡4倍觀察結果。以 0 μg/ml為Control組；DMSO (劑量為0.50%)為Vehicle49
圖14. 以不同劑量魚針草萃取物處理24 hr對抑制FaDu細胞移行之效應。以倒立式相位差顯微鏡10倍觀察結果。以 0 μg/ml為Control組；DMSO (劑量為0.50%)為Vehicle圖目錄。
圖15. 以不同劑量魚針草萃取物處理24 hr對抑制FaDu細胞移行之效應。使用0μg/ml為Control組；DMSO (劑量為0.50%)為Vehicle。圖表數值是將染色的insert退染後，在用ELISA定量溶液的顏色50
圖16. 以不同劑量魚針草萃取物處理24 hr對抑制FaDu細胞入侵之效應。以倒立式相位差顯微鏡4倍觀察結果。以 0 μg/ml為Control組；DMSO (劑量為0.50%)Vehicle52
圖17. 以不同劑量魚針草萃取物處理24 hr對抑制FaDu細胞入侵之效應。以倒立式相位差顯微鏡10倍觀察結果。以 0 μg/ml為Control組；DMSO (劑量為0.50%)為Vehicle52
圖18. 以不同劑量魚針草萃取物處理24 hr對抑制FaDu細胞入侵之效應。使用 0μg/ml為Control組；DMSO (劑量為0.50%)為Vehicle。圖表數值是將染色的insert退染後，在用ELISA定量溶液的顏色。53
圖19. 以不同劑量魚針草萃取物對FaDu癌細胞處理24後，(a)細胞內訊息因子的表現量(MAPK pathway)，以β-actin作為loading control；(b)細胞核內的訊息因子NF-κB表現量/PARP的比值，PARP為核內蛋白表現量的loading control。55
圖20. 不同劑量魚針草萃取物對FaDu癌細胞處理24後對上清液中MMP-2及MMP-9含量之影響。使用R&D Systems Quantikine的MMP-2 & MMP-9ELISA kit 進行MMP-2及MMP-9的含量測定。57
圖21. 以不同劑量魚針草萃取物對FaDu癌細胞處理24hr後的濃縮培養液(濃度為25 μg/ml)，對於含gelatin的SDS page的侵蝕作用，藉此判定抑制MMP-2及MMP-9活性的效果。58
表目錄
表1. 民國98年十大死因2
表2. 民國98年全國男性癌症十大死因2
表3. 民國98年全國女性癌症十大死因3
表4. 魚針草各萃取分層重量及含水率34
表5. 魚針草萃取物對A549不同時間之毒殺效果35
表6. 魚針草萃取物對FaDu及MRC-5細胞處理24 hr之毒殺效果35

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