臺灣博碩士論文加值系統

白英是一種傳統的中草藥，通常用來消炎、抗癌。一些研究表明，白英具有抗腫瘤特性，但機制不為人所熟知。因此，白英粗抽物極性分層對人類口腔癌細胞（HSC-3/SAS/CAL27）反應進行了研究。人類口腔癌細胞（HSC-3/SAS/CAL27）作用在不同濃度，不同的作用時間後，檢測細胞存活率，細胞形態變化，細胞週期阻滯，凋亡，活性氧（ROS），粒線體膜電位，細胞週期和細胞凋亡相關蛋白表現。結果顯示，氯仿分層產物作用後明顯降低了三株細胞的存活率，並伴隨著細胞型態的改變。經由計算，SLEC (Solanum lyratum extracts by CHCl3)作用於三株細胞株可得出IC50約略為40 ?慊/ml (SLEC 40)，並以此濃度作進一步的探討。而結果指出SLEC作用後可促使三株口腔癌細胞(HSC-3/SAS/CAL27)的細胞週期停滯於G0/G1期和產生細胞凋亡，也可從結果發現三株細胞對於藥物具有劑量依賴性。從DAPI染色可證實SLEC誘發三株口腔癌細胞(HSC-3/SAS/CAL27)產生細胞凋亡。而從流式細胞儀檢測中可顯示SLEC可促使細胞產生活性氧化物質 (在SAS細胞無顯示)，並促使細胞的粒線體膜電位降低。在西方點墨法分析中指出SLEC 40促使Bax蛋白表現量上升與Bcl-XL的表現量下降，而使細胞粒線體膜電位下降，產生cytochrome c，促使下游caspase -9 活化，最後促使細胞產生細胞凋亡。SLEC 40亦會促使p16、p21的表現量增加，而使得細胞週期在G0/G1停滯。從以上結果發現，SLEC在未來有可能會被使用當作一種抗口腔癌的治療藥物。

Solanum lyratum Thunb. is a traditional herbal medicine for diminishing inflammation, anti-oncogenesis and anti-cancer. Several studies have indicated that Solanum lyratum Thunb. have anti-tumor properties, but the mechanism is not well known. Therefore, the effect of the polar fraction of extracts from Solanum lyratum on human oral cancer cells (HSC-3/SAS/CAL27) were investigated. The cell viability, morphological changes of the cells, cell cycle arrest, apoptosis, reactive oxygen species (ROS), mitochondrial membrane potential and apoptosis-associated protein levels were examined in human oral cancer cells (HSC-3/SAS/CAL27) cells after exposure to various concentrations of polar fractions of SLE (Solanum lyratum extract). The results indicated that polar fraction extracts by CHCl3 decreased the percentage of viability in three kind of oral cancer cells accompanied by morphological changes. The IC50 of SLEC (Solanum lyratum extracts by CHCl3) was 40 ?慊/ml (SLEC 40) and this concentration was used for further investigations. SLEC induced G0/G1 phase arrest and causes apoptosis in the oral cancer cells (HSC-3/SAS/CAL27) at a dose dependent manner. DAPI staining confirmed that SLEC induced apoptosis in oral cancer cells (HSC-3/SAS/CAL27). Flow cytometric analysis also showed that SLEC causes or enhanced ROS production (but not in SAS) and decreased the mitochondrial membrane potential. Western blot analysis indicated that SLEC 40 increased Bax levels and decreased Bcl-XL levels, which caused the loss of mitochondrial membrane potential followed, and cytochrome c release and caspase-9 activation, finally led to apoptosis. SLEC 40 also up-regutaled p16, p21, thus causing G0/G1 phase arrest. These findings suggest that SLEC might be used as an oral cancer therapeutic agent in the future.

總目錄
總目錄 ………………………………………………………………. i
圖目錄 ………………………………………………………………. v
表目錄 ………………………………………………………………. vii
致謝 …………………………………………………………………. viii
縮寫表 ………………………………………………………………. ix
中文摘要 ……………………………………………………………. x
英文摘要 ……………………………………………………………. xii
前言 …………………………………………………………………. 1
文獻回顧 .…………………………………………………………… 4
一 癌症成因 ……………………………………………………… 4
二 台灣口腔癌的流行病學 ……………………………………… 6
三 白英抗腫瘤的藥理作用 ……………………………………… 9
四 細胞死亡 (Cell death)之路徑 ………………………………… 11
五 活性氧物ROS (Reactive Oxygen Species)及抗氧化酵素 …... 14
研究材料與方法 ……………………………………………………. 19
一 實驗材料 ………………………………………………………. 19
細胞株 ……………………………………………………… 19
藥物試劑 ……………………………………………………. 19
儀器設備與器材 ……………………………………………. 22
二 實驗目的 ………………………………………………………… 23
三 實驗設計 ………………………………………………………… 23
四 實驗方法 ………………………………………………………… 24
1 配製藥物 ……………………………………………………. 24
2 Cell culture (細胞培養) ……………………………………. 24
3 細胞數目測定 ……………………………………………… 25
4 流式細胞儀測定分析………………………………………… 25
5 DNA內螢光染色 (Fluorescence staining) ………………… 37
6 西方點墨法（Western Blotting）………………………….. 39
7 免疫螢光染色 (Immunofluorescence Staining) ………….. 41
8 統計分析 (Statistics analysis) ……………………………… 42
研究結果 ……………………………………………………………. 43
一 白英粗抽物各分層對人類口腔癌細胞HSC-3/SAS/CAL27細胞毒性的探討………………………………………………
43
二 白英粗抽物氯仿分層（SLEC）對口腔癌細胞HSC-3/SAS/CAL27存活率（Cell Viability）的探討…………
46
三 白英粗抽物氯仿分層（SLEC）對口腔癌細胞HSC-3/SAS/CAL27形態的影響………………………………
49

四
探討白英粗抽物氯仿分層（SLEC）對口腔癌細胞HSC-3/SAS/CAL27細胞週期之影響 ……………………….

51
五 探討白英粗抽物氯仿分層（SLEC）對口腔癌細胞HSC-3/SAS/CAL27細胞週期相關蛋白之影響………………
55
六 觀察白英粗抽物氯仿分層（SLEC）對口腔癌細胞HSC-3/SAS/CAL27 DNA損傷之情形………………………
56
七 探討白英粗抽物氯仿分層（SLEC）對口腔癌細胞 HSC-3/SAS/CAL27 活性氧化物質（ROS）釋放的影響……
62
八 探討白英粗抽物氯仿分層（SLEC）對口腔癌細胞 HSC-3/SAS/CAL27 細胞抗氧化相關蛋白之影響…………..
65
九 探討白英粗抽物氯仿分層（SLEC）對口腔癌細胞 HSC-3/SAS/CAL27 產生一氧化氮（Nitric oxide, NO）影響
66
十 探討白英粗抽物氯仿分層（SLEC）對口腔癌細胞 HSC-3/SAS/CAL27 一氧化氮釋放相關蛋白之影響……….
68
十一 探討白英粗抽物氯仿分層（SLEC）對口腔癌細胞HSC-3/SAS/CAL27鈣離子（Ca2+）釋放之影響 ………….
69
十二 探討白英粗抽物氯仿分層（SLEC）對口腔癌細胞HSC-3/SAS/CAL27細胞凋亡內質網壓力路徑相關蛋白之表現 ……………………………………………………………..

72
十三 探討白英粗抽物氯仿分層（SLEC）對口腔癌細胞HSC-3/SAS/CAL27粒線體膜電位之影響 …………………
73
十四
探討白英粗抽物氯仿分層（SLEC）對口腔癌細胞HSC-3/SAS/CAL27細胞凋亡粒線體路徑相關蛋白之表現影響 ……………………………………………………………..

76
十五 探討白英粗抽物氯仿分層（SLEC）對口腔癌細胞HSC-3/SAS/CAL27細胞凋亡相關蛋白變化之影響 ………
77
十六 利用免疫螢光染色法探討白英粗抽物氯仿分層（SLEC）對口腔癌細胞HSC-3/SAS/CAL27蛋白轉位（translocatiom）表現……………………………………………………………..

78
十七 探討白英粗抽物氯仿分層（SLEC）對口腔癌細胞HSC-3/SAS/CAL27之 capase-3/-8/-9活性之影響 …………
80
十八 探討caspase-3/-8/-9抑制劑對口腔癌細胞HSC-3/SAS/CAL27存活率變化之影響 ……………………
84
結果討論 ……………………………………………………………... 87
實驗結論 …………………………………………………………….. 93
參考文獻 …………………………………………………………….. 95

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