臺灣博碩士論文加值系統

中文摘要
在台灣，口腔癌的致死率佔國人癌症死因之第五名。Oroxylin A (Oro-A) 屬黃芩根部的萃取物，具有解熱、鎮痛、抗發炎、及抗癌的功效。由於癌細胞的移行在腫瘤轉移的機制扮演重要的角色，因此，本研究主要目的為暸解Oro-A對口腔癌移行之影響及其分子機制。利用細胞存活率分析，我們發現低劑量Oro-A並不影響口腔癌細胞的存活率。細胞傷口癒合實驗發現低劑量Oro-A可以有效抑制口腔癌細胞的移行作用。長時間 (10代繼代) 低濃度 (20 M) 處理Oro-A抑制口腔癌細胞的移行作用更為顯著藥效更佳。我們以cDNA microarray、Ingenuity軟體分析長時間Oro-A處理下基因表現變異及進一步以QPCR確認其中與癌細胞移行相關基因表現。QPCR結果發現長時間Oro-A處理可降低CCL2、S-100A9、CSF-2、THBS-1 mRNA 表現。我們進一步發現長時間Oro-A處理也可以有效的降低soluble CCL2蛋白質及其下游MAPK與MMPs的表現。最後，我們以實驗性動物體內腫瘤轉移模式測試長時間Oro-A處理對體內轉移之影響。總結本研究的實驗成果推論長時間Oro-A處理可透過CCL2 -MAPK-MMPs路徑抑制口腔癌細胞移行。

Oral cancer is rated 5th in cancer-related death in Taiwan in 2014. Oroxylin A (Oro-A), a main bioactive flavonoid extracted from Scutellaria radix, is prescribed having analgesic, antipyretic , anti-inflammation, and anti-cancer. This study aims to investigate the anti-migration effect of Oro-A and its underlying mechanisms in oral cancer cells. By using cell viability assay, we found that short term low dose of Oro-A did not affect the viability of human oral cancer cells. By using wound healing assay, we found that short term 3 days Oro-A (20 M) exposure significantly inhibited cell migration. In addition, long-term exposure 30 days of Oro-A, which had no cytotoxic effect, significantly inhibited more cell migration than that in 24 hours Oro-A-exposured oral cancer cells. We further used cDNA microarray and ingenuity software to identify the gene profile changes in long term Oro-A-exposured oral cancer cells. The mRNA of CCL2, S-100A9, LCN-2, THBS-1, CSF-2, MMP-2 and MMP-9 were reduced in long term Oro-A exposed oral cancer cells. In addition, the concentration of secreted CCL2 level was reduced by long term 30 days Oro-A treatment. The protein levels down regulator of CCL2, including p-ERK1/2, p-JNK1/2/3, MMP-2 and MMP-9, were significantly reduced in long term Oro-A treatment. Finally, we adopted experimental in vivo metastasis animal to examine the effect of Oro-A on in vivo cancer metastasis. The result indicated that long term in vitro Oro-A exposure had no effect on in vivo metastasis. Taken together, our results suggest that Oro-A has the therapeutic potential against human oral cancer cells migration.

目錄
壹、 中文摘要----------------------------------------------------------9
貳、 英文摘要----------------------------------------------------------10
參、 研究背景----------------------------------------------------------12
●口腔癌簡介-----------------------------------------------------------12
●癌細胞轉移的進程--------------------------------------------------13
●Oro-A的背景與抗癌研究-----------------------------------------14
肆、研究動機與目的----------------------------------------------15
伍、實驗材料與方法----------------------------------------------16
一、材料--------------------------------------------------------------16
二、實驗方法---------------------------------------------------------18
1. 細胞培養、計數--------------------------------------------18
2. 藥物處理模式-----------------------------------------------18
3. 細胞存活率測試--------------------------------------------19
4. 細胞傷口癒合試驗-----------------------------------------20
5. 以Trypan blue staining assay計算口腔癌細胞的生長速度------------------------------------------------------------------21
6. 細胞群落分析--------------------------------------------22
7. 西方墨點法------------------------------------------------22
8. cDNA microarray analysis--------------------------------23
9. 即時定量聚合酶連鎖反應------------------------------24
10. Quantification of CCL2 by ELISA analysis-----------25
11. 體內腫瘤轉移動物實驗--------------------------------26
12. 實驗數據統計分析--------------------------------------26
陸、 實驗結果---------------------------------------------------------28
一、 短期Oro-A處理對於口腔癌細胞毒性及移行的影響----------28
二、 長期Oro-A處理對於口腔癌細胞生長及移行的影響----------28
三、 長期Oro-A處理對於口腔癌細胞基因表現影響-----------------29
四、 長時間Oro-A處理對於口腔癌細胞移行相關基因的表現量分析-----------------------------------------------------------------------------30
五、 長期Oro-A處理可顯著降低CCL2的medium 濃度與蛋白質表現量------------------------------------------------------------------------30
六、 長期Oro-A處理可顯著降低MAPK 與MMP相關蛋白表現------------------------------------------------------------------------------31
七、 長期Oro-A處理對於口腔癌細胞體內腫瘤轉移的影響-------32
柒、 討論-----------------------------------------------------------------33
捌、 結論-----------------------------------------------------------------40
玖、 參考文獻---------------------------------------------------------62
表格目錄
Table 1. Primer sequences for quantitative real-time polymerase chain reaction assays (qPCR)--------------------------------------------------------41
Table 2. cDNA microarray data 分析-------------------------------------42
Table 3. 腫瘤轉移的位置--------------------------------------------------45

圖目錄
Figure 1. Oro-A對於口腔癌細胞的存活率影響與移行抑制的情況-----------------------------------------------------------------------------------47
Figure 2. Oro-A長期處理對於口腔癌細胞群落之形成與生長速率的影響--------------------------------------------------------------------------------49
Figure 3. 長時間Oro-A處理對於QPCR的表現量分析-------------54
Figure 4. 長期處理Oro-A對於CCL2的影響--------------------------55
Figure 5. 長期處理Oro-A對於MAPK蛋白質表現量的影響------56
Figure 6. 長期處理Oro-A對於MMP相關蛋白質與qPCR表現量的影響--------------------------------------------------------------------------------58
Figure 7. Oro-A對於口腔癌細胞腫瘤生長的影響---------------------60
Figure 8. Oro-A抑制口腔癌細胞移行作用的機制分析圖------------61

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