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研究生:彭詩媛
研究生(外文):Shih-Yuan Peng
論文名稱:BI-D1870之抗口腔癌細胞活性
論文名稱(外文):Antitumor Effects of BI-D1870 on Oral Cancer Cells
指導教授:翁靖如
指導教授(外文):Jing-Ru Weng 翁靖如
學位類別:碩士
校院名稱:中國醫藥大學
系所名稱:生物科技學系碩士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:72
中文關鍵詞:口腔癌Ribosomal S6 kinaseBI-D1870設定性凋亡
外文關鍵詞:oral cancerRibosomal S6 kinaseBI-D1870apoptosis
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Mitogen-Activated protein kinase (MAPK) 活化後,進行一連串複雜的細胞反應,如細胞增生、分化及細胞凋亡等。 Ribosomal S6 kinase (RSK),在MAPK 路徑中扮演重要角色,經由磷酸化調控許多細胞質與細胞核蛋白質的功能,亦會調節一些轉錄因子,使得細胞增生、分化或存活等作用,並有文獻指出 RSK 在乳癌和前列腺癌中亦會大量表現。文獻報告指出 BI-D1870為RSK 抑制劑之一, 顯示對 RSK 有專一性的細胞傳遞之抑制活性,且為 ATP 競爭型抑制劑。本論文探討 BI-D1870 對於口腔癌細胞的生長,細胞週期調控及凋亡因子的影響,利用 MTT assay 和 Cell counting 測試 BI-D1870對於口腔癌細胞 (SCC2095, SCC4, SCC9, Ca922, and HSC3) 及正常人類口腔上皮細胞 (Normal human keratinocytes) 的生長抑制。並利用流式細胞儀偵測口腔癌細胞的細胞週期、細胞凋亡、以及活性氧基群的表現,同時也利用 DAPI staining 偵測細胞核質濃縮現象。結果顯示 BI-D1870 會抑制口腔癌細胞的生長,對於口腔癌細胞,SCC4 cells 最具毒殺效果,IC50為1.7 ?嵱,並造成細胞週期停滯在 G2/M 期,進而造成細胞凋亡。同時也會促使粒線體凋亡路徑及細胞週期相關蛋白的活化,除此之外,BI-D1870 亦會抑制 RSK 之受質與細胞增生相關的蛋白表現量。

Mitogen-activated protein kinase (MAPK) executes complex cellular programs, like proliferation, differentiation and apoptosis. Ribosomal S6 kinase (RSK), a key player in MAPK pathway, modulates the activities of several cytoplasmic and nuclear proteins via phosphorylation. It also regulated some transcription factors, lead to the cell proliferation, differentiation and survival. And there are some papers to show that RSK was overexpression in breast cancer and prostate cancer. It has been reported recently, that BI-D1870, one of RSK inhibitors, showed the specific cell-permeant inhibitory activity of RSK, which acts as an ATP competitive inhibitor. In this study, we explore the effects of BI-D1870 for cell growth, cell cycle and apoptosis on oral cancer cells. We used MTT assay and Cell counting test to detect cell survival in oral cancer cells (SCC2095, SCC4, SCC9, Ca922, and HSC3) and normal human keratinocytes. Then flow cytometry was used to detect cell cycle and apoptosis, and the activity of reactive oxygen species in oral cancer cells, DAPI staining was also used to detect nuclear concentration. Results show that BI-D1870 inhibits oral cancer cells growth inducing cell cycle arrest in the G2/M phase, and causes apoptosis. In addition, BI-D1870 also inhibits RSK and cell proliferation-related protein expression.

中文摘要 I

英文摘要 II

誌謝 III

目錄 IV

圖目錄 VIII

表目錄 X

第一章 前言 1
第二章 緒論 4
第一節 實驗藥物:BI-D1870 4
第二節 實驗癌症之探討 5
一、癌症 5
二、口腔癌 5
三、口腔癌成因 6
四、臨床症狀 6
五、口腔癌的治療 7
六、研究動機 7
第三節 實驗目的與設計流程 8
一、實驗目的 8
二、實驗設計流程 8
第三章 研究材料與方法 9
第一節 實驗材料 9
一、細胞株 9
二、實驗藥物 9
三、藥品試劑 10
四、儀器設備 13
第二節 實驗方法 15
一、 實驗藥物配置 15
二、細胞培養 16
(一) 配置細胞培養基 16
(二) 細胞株培養條件 16
(三) 解凍細胞 16
(四) 冷凍細胞 17
三、細胞存活率試驗 (MTT Assay) 18
四、流式細胞儀分析 (Flow Cytometry) 20
(一) 細胞週期檢測 (PI staining) 20
(二) 細胞凋亡檢測 (Annexin V / PI staining) 22
(三) 活性氧的測定 (Reactive Oxygen Species assay) 24
五、DAPI assay (DAPI staning)
25
六、西方墨點法 (Western blotting) 26
(一) 細胞總蛋白的抽取 26
(二) 細胞濃度的測定 26
七、統計分析 32
第四章 實驗結果 33
第一節 BI-D1870對口腔癌細胞 (SCC2095, SCC4, SCC9, Ca922, and HSC3) 及正常人類口腔上皮細胞(Normal human keratinocytes)的存活率及型態影響之探討

33
第二節 BI-D1870 對口腔癌細胞 (SCC2095, SCC4, SCC9, Ca922, and HSC3) 的存活數目之探討
35
第三節 BI-D1870對口腔癌細胞SCC4的細胞週期及相關蛋白質表現量影響之探討
36
第四節 BI-D1870對口腔癌細胞SCC4的細胞凋亡引發及相關蛋白質表現量影響之探討
39
第五節 BI-D1870對口腔癌細胞SCC4的ROS引發及相關蛋白質表現量影響之探討
42
第六節 BI-D1870對口腔癌細胞SCC4的胞內蛋白質表現量變化之影響 43
一、探討BI-D1870對RSK上游之相關蛋白質表現量影響 43
二、探討BI-D1870對RSK受質之相關蛋白質表現量影響 43
三、探討BI-D1870對NF-?羠路徑之相關蛋白質表現量影響 43
第七節 實驗結果圖表 45
第五章 討論 62
第六章 結論 66
第七章 參考文獻 67
















圖目錄
Figure 1. 口腔解剖圖 5
Figure 2. 實驗設計流程 8
Figure 3. The chemical structure of BI-D1870. 9
Figure 4. MTT會被代謝轉變為Formazan 18
Figure 5. Cell cycle 分析圖表範例 21
Figure 6. 細胞調亡PS從細胞膜內層外翻至外層圖 23
Figure 7. Cell apoptosis 分析圖表範例 23
Figure 8. 西方墨點法-轉漬夾的內部組成 31
Figure 9. 細胞計數器 35
Figure 10. 細胞週期 37
Figure 11. 細胞內和細胞外的凋亡路徑圖 40
Figure 12. The antiproliferative effect of BI-D1870 at the indicated concentrations on SCC2095, SCC4, and SCC9 oral cancer cells.
45
Figure 13. The antiproliferative effect of BI-D1870 at the indicated concentrations on Ca922 and HSC3 oral cancer cells and normal human keratinocytes (NHOK).

46
Figure 14. The antiproliferative effect of BI-D1870 at the indicated concentrations in SCC2095, SCC4, and SCC9 oral cancer cells.
48
Figure 15. The distribution of cell cycle of SCC4 cells after treatment with BI-D1870 for 48h.

49
Figure 16. The distribution of cell cycle of SCC4 cells after treatment with BI-D1870 for 48h.
50
Figure 17. Effects of BI-D1870 on cell cycle-related biomarkers in SCC4 oral cancer cells.
51
Figure 18. Flow cytometry analysis of apoptosis on SCC4 cells after treatment with BI-D1870 for 48h.
52
Figure 19. Flow cytometry analysis of apoptosis on SCC4 cells after treatment with BI-D1870 for 48h.
53
Figure 20. Effect of BI-D1870 on SCC4 cells in different concentration after treating for 48h by DAPI assay. 54
Figure 21. Effects of BI-D1870 on apoptosis-related biomarkers in SCC4 oral cancer cells.
55
Figure 22. Effects of BI-D1870 on Bcl-2 family - related proteins in SCC4 oral cancer cells.
56
Figure 23. The distribution of ROS percentages of SCC4 cells after treatment with BI-D1870 for 48h.
57
Figure 24. Effects of BI-D1870 on ER-stress-related biomarkers in SCC4 oral cancer cells.
58
Figure 25. Effects of BI-D1870 on AKT pathway-related proteins in SCC4 oral cancer cells.
59
Figure 26. Effects of BI-D1870 on RSK-related proteins in SCC4 oral cancer cells.
60
Figure 27. Effects of BI-D1870 on NF-κ B pathway-related proteins in SCC4 oral cancer cells.
61
Figure 28. Antitumor effects of BI-D1870 in oral cancer SCC4 cells. 66


表目錄
Table 1. SDS-PAGE下層膠(separation gel)之配製及組成 27
Table 2. SDS-PAGE上層膠(stacking gel)之配製及組成 27
Table 3. 電泳緩衝液(running buffer;1.5 M Tris-HCl, pH 8.8)之組成 28
Table 4. Stacking buffer(0.5 M Tris-HCl, pH 6.8)之組成 28
Table 5. 轉印緩衝液(transfer buffer)之組成 30
Table 6. PBS-tween 20之組成 31
Table 7. Bcl-2 family 分類表 41
Table 8. The IC50 values of BI-D1870 on various oral cancer cell lines and normal human keratinocytes. 47
Table 9. The percentage of SCC4 cells in each phase regulated by BI-D1870. 50
Table 10. The percentage of SCC4 cells in each phase regulated by BI-D1870. 53


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