Protoapigenone (AN30)化合物是由植物大金星蕨類Thelypteris torresiana分離之萃取物。AN30是一種黃酮類化合物，在我們先前研究中發現AN30能抑制肺癌細胞凋亡，也有文獻指出AN30，會誘導細胞啟動自噬作用使細胞死亡，近年來的研究中有越來越多的證明，選擇性啟動細胞凋亡確實增加了癌症化療的效力。AN30在目前已知研究結果中確定其能使細胞啟動細胞凋亡及自噬作用，但是AN30在cell death中之作用機制仍不清楚。自噬作用己被認為與抗藥性相關，我們發現AN30對細胞造成細胞凋亡時也發生自噬作用。將細胞starvation後再處理AN30結果顯示細胞經starvation後AN30對細胞的毒性明顯降低(Ca9-22處理AN30：1.5 µg/ml之細胞存活率為64% 而經starvation後增加至139%)。因此我們利用了自噬作用之指標Acridine orange staining、Western blotting及自噬作用抑制劑chloroquine來探討starvation與細胞毒性及自噬作用的關係。實驗結果中發現，經starvation後細胞產生自噬作用，再將細胞共同處理AN30及自噬抑制劑chloroquine則明顯降低細胞毒性(Ca9-22共同處理AN30：1.5 µg/ml及CQ12.5 µM 24小時，其存活率為34%，而經starvation後再將細胞共同處理AN30：1.5 µg/ml及CQ12.5 µM 24小時，其存活率增加至61%)，並伴隨著AO染色升高。然而AN30共同處理自噬作用抑制劑chloroquine，並無法發現有明顯的抑制細胞自噬作用及升高細胞毒性。我們先前研究中證實AN30會使細胞產生ROS，未來可先將細胞starvation後再處理AN30確定是否會使降低ROS的產生，並進一步觀察自噬作用指標LC3- II的變化。

AN30 is extracted from stem and leaves of Thelypteris torresiana. Recently, select killing cancer cells through apoptosis and other mechanisms are proved to increase the efficacy of chemotherapy. Our previous studies indicate that AN30 can select kill oral cancer cells, however the mechanism is unclear. Autophagy is associated with chemotherapy drug. We found AN30 can induce apoptosis and autophagy. When oral cancer was pretreated with starvation, the AN30 cell toxicity is significantly reduced (cell survival rate of Ca9-22, from 64% in 1.5 µg/ml AN30 to 139% in starvation pretreatmen). Then we use autophagy maker acridine orange staining, LC3 in western blot analysis, and autophagy inhibitor chloroquine to investigate the relation of starvation, cell toxicity and autophagy. Our experimental results show the starvation induce autophagy, then suppress AN30 cell toxicity (24 hr cell survival rate of Ca9-22, from 64% in 1.5 µg/ml AN30 to 139% in starvation pretreatment), with high AO fluorescence expression in oral cancer cells, though the LC3-II level . But co-treat AN30 and autophagy inhibitor chloroquine cannot inhibit the starvation induced AN30 resistance in oral cancer cells (24 hr cell survival rate of Ca9-22, from 34% in 1.5 µg/ml AN30 and 12.5 CQ, to 61% in starvation pretreatment compared to 65% without CQ co-treatment). Previous studies indicate that AN30 can increase cellular ROS levels. Our future work will focus on the mechanism of starvation induced drug resistance which may associate with autophagy and ROS scavenge.

論文審定書 i
致謝 ii
摘要 iii
Abstract iv
Abbreviation v
目錄 vi
第一章、前言 1
1.1口腔癌背景介紹 1
1.2口腔癌成因 1
1.3口腔癌症狀 1
1.4口腔癌分期及其五年存活率 2
1.5口腔癌治療 3
1.6 Protoapigenone (AN30) 4
1.7 細胞死亡 5
1.8 自噬作用 ( autophagy ) 5
1.9 在口腔癌 co-treatment藥物 7
1.10 細胞凋亡 ( Apoptosis ) 7
第二章、實驗目的 9
第三章、實驗流程 10
第四章、實驗方法 11
4.1 細胞株 11
4.2 培養材料 11
4.3 細胞繼代培養 11
4.4 細胞解凍 12
4.5 MTT assay 13
4.6 Flow cytometry 13
4.7 蛋白質製備 14
4.8 蛋白質濃度測定 15
4.9 Acridine Orange stain 16
4.10 Image J 影像分析 16
4.11西方點墨法(Western blot) 16
4.12統計分析 (Statistical Analysis) 20
第五章、實驗結果 21
5.1 對口腔癌細胞處理天然藥物 AN30 使其存活率下降 21
5.2 對口腔癌細胞處理天然藥物 AN30 使細胞之 autophagy 增加 21
5.3 Starvation 使口腔癌細胞產生 autophagy ，促進口腔癌細胞對 AN30 之抗藥性 22
5.4 將細胞共同處理 autophagy 抑制劑 CQ 及天然藥物 AN30 ，測試口腔癌細胞存活率 23
5.5 在starvation後將細胞共同處理 autophagy 抑制劑 CQ 及天然藥物 AN30 ，則口腔癌細胞存活率比未處理starvation之實驗組增加 24
第六章、結果討論 27
6.1天然藥物 AN30 對口腔細胞存活率與自噬作用的關係 27
6.2藉由starvation誘導細胞自噬作用來探討細胞存活率和自噬作用之關係 27
6.3 AN30、CQ cotreat的作用機制 27
6.4 ROS 可能參與細胞調控之 apoptpsis 、 autophagy 發生 28
第七章、參考文獻 30
第八章、圖表 34
Figure 1. AN30 induce cell death in oral cancer cells. 34
Figure 2. AN30 induce autophagy in oral cancer cells. 37
Figure 3. Starvation supress AN30-induce cell death in oral cancer cells. 38
Figure 4. AN30 induce autophagy after starvation in oral cancer cells. 41
Figure 5. Western blot of LC3-I and LC3-II with AN30 treatment in oral cancer cells. 42
Figure 6. Cell viability after co-treat with AN30 and CQ in oral cancer cells. 45
Figure 7. AN30 co-treat with CQ induce autophagy in oral cancer cells. 50
Figure 8. Cell viability after preteat starvation following co-treat with AN30 and CQ in oral cancer cells. 52
Figure 9. AO staining of oral cancer cell after starvation pretreat and AN30 CQ co-treat. 57
Figure 10. LC3-I and LC3-II western blot of starvation pretreatment following AN30 and CQ co-treatment in oral cancer cells. 60
第九章、附錄 61
9.1 Flow cytometry analysis：PI stain of Ca9-22 treated with 0µg/ml AN30 for 24 hr. 61
9.2 Flow cytometry analysis：PI stain of Ca9-22 treated with 1µg/ml AN30 for 24 hr. 61
9.3 Flow cytometry analysis：PI stain of HGF treated with 0µg/ml AN30 for 24 hr. 62
9.4 Flow cytometry analysis：PI stain of HGF treated with 1µg/ml AN30 for 24 hr. 62
9.5 LC3-I and LC3-II western blot of Ca9-22 treated with CQ. 63
9.6 LC3-I and LC3-II western blot of HGF treated with CQ. 63
9.7 CQ induce cell death in Ca9-22. 64
9.8 CQ induce cell death in HGF. 64
9.9 CQ induce cell death in Ca9-22 pretreated with starvation. 65
9.10 CQ induce cell death in HGF pretreated with starvation. 65

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