臺灣博碩士論文加值系統

嚼食檳榔是受歡迎的口腔嗜好之一，估計全球約有二到六億的嚼食人口。流行病學的研究發現嚼食檳榔與口腔癌間具有強烈的關連性。在台灣，檳榔嚼塊通常是由新鮮的檳榔子，紅灰或白灰，再加上荖花或荖葉所組成。許多的研究都指出檳榔子具有致癌性與基因毒性；然而，截至目前為止，對於檳榔子的致癌機轉仍是不完全清楚。從本篇論文中發現隨著濃度與時間的遞增，檳榔子萃取物會抑制CHO-K1 細胞的生長與分裂。經過二十四小時的處理後，濃度高於400μg/ml 的檳榔子萃取物會增加細胞內過氧化氫的含量。從細胞質分裂阻斷之微核分析法中發現檳榔子萃取物會誘導微核的生成且伴隨著細胞內過氧化氫濃度的提升。當以400~800μg/ml 的濃度作用二十四小時，發現會造成細胞週期停滯於G2/M phase；當作用時間拉長，發現檳榔子萃取物會造成細胞死亡。有趣的是，我們還觀察到檳榔子萃取物作用二十四小時後的細胞會出現細胞質分裂缺損，且雙核細胞的比例有明顯增多的現象。相似的濃度下，發現檳榔子萃取物會擾亂 actin filament 的分佈。而抗氧化劑Catalase (2000U/ml)與檳榔子萃取物(800μg/ml)共同作用下的細胞，雙核細胞的生成會受到明顯的抑制，這也意味著檳榔子萃取物所造成的細胞質分裂缺損與活性氧的生成有關。另外，長時間的作用下多倍體，多核或微核細胞之數量有明顯的增多，同時也發現數量異常之中心體。除此之外，我們還發現2mM 之咖啡因會加成檳榔子萃取物之細胞毒性，且可有效的減少多倍體細胞的數量。故從本實驗的結果，我們認為檳榔子萃取物會增加微核頻率，誘導細胞週期停滯於G2/M phase，造成細胞質分裂缺損與多倍體細胞的增多；而這些現象可能與活性氧的生成，actin filament 的異常與中心體的變異有關。

Betel quid (BQ) chewing is the fourth most popular oral habit. There are about 200 - 600 million BQ chewers in the world. Epidemiological evidences have found the strong association between BQ chewing and oral cancer. In Taiwan, BQ consists of fresh areca nut (AN) and lime with or without inflorescence piper betle or betle leaf. Many studies have demonstrated that AN exerts mutagenicity and genotoxicity. However, the precise toxic mechanisms responsible for AN-induced carcinogenesis are not fully clear. In this thesis, we found that AN extract (ANE) inhibited the growth and proliferation of CHO-K1 cells in a concentration- and time-dependent manner. After 24-h of exposure, ANE increased the intracellular H2O2 production at concentrations higher than 400 µg/ml. By using cytokinesis-block micronucleus assay, ANE induced micronuclei formation in CHO-K1 cells, accompanied by an increase in intracellular H2O2. Incubation of CHO-K1 cells with ANE (400~800 µg/ml) for 24-h caused G2/M cell cycle arrest and prolonged exposure to 800 µg/ml of ANE induced cell death. Interestingly, we observed that ANE itself caused cytokinesis failure and increased the frequency of binucleated cells following 24-h exposure. At similar concentrations, ANE induced actin filament disorganization. Co-incubation of cells to catalase (2000 U/ml) and ANE (800 µg/ml) reduced the binucleated cells generation, indicating that ANE-induced cytokinesis failure was associated with ROS production. After prolonged exposure to ANE, the accumulation of hyperploid cells, micronucleated or multinucleated cells were noted concomitantly with centrosome amplification. In addition, we found that co-exposure of cells to caffeine (2 mM) and ANE (800 µg/ml) resulted in additive or synergistic cytotoxicity, accompanied by a decrease in hyperploid cells. In summary, our results suggest that ANE increases micronuclei frequency, induces cell cycle arrest at G2/M phase, causes cytokinesis failure and accumulates hyperploid cells. The events are possibly associated with ROS production, actin filament disorganization and centrosome aberration.

中文摘要---------------------------------------------------------------------I
英文摘要--------------------------------------------------------------------II
目錄-----------------------------------------------------------------------III
圖次-------------------------------------------------------------------------V
附錄-----------------------------------------------------------------------VII

第一章 緒論-----------------------------------------------------------------1
1.1. 口腔黏膜病變與嚼食檳榔間的關係------------------------------------1
1.2. 微核(Micronucleus, MN)----------------------------------------------3
1.3. 細胞質分裂缺損(cytokinesis failure)與癌症形成間的關連性----------------5
1.4. 檳榔子萃取物對細胞週期之影響-------------------------------------7
1.5. 咖啡因對細胞週期與細胞死亡的影響------------------------------9

實驗動機與目的---------------------------------------------------------12

第二章 實驗材料方法-------------------------------13
2.0. 實驗材料與藥品之來源---------------------------------------------13
2.1. 檳榔子之水萃取---------------------------------------------------13
2.2. 細胞株與細胞培養-------------------------------------------------14
2.3. 細胞存活率試驗--------------------------------14
2.3.1. 錐蟲藍排除法(Trypan blue exclusion assay)-------------------14
2.3.2. 細胞群落形成效率分析(Colony formation efficiency, CFE)---------14
2.4.細胞質分裂阻斷之微核分析(Cytokinesis-block micronucleus assay)---------15
2.5.利用流式細胞技術分析細胞內活性氧含量-----------------------------16
2.6. 利用流式細胞儀分析細胞週期之變化---------------------------------17
2.7. 免疫螢光顯微鏡 (Immunofluorescence microscopy)----17
2.7.1. F-actin之染色---------------------------------------------17
2.7.2. γ-tubulin之染色------------------------------------------18
2.8. 統計分析---------------------------------------------------------18

第三章 實驗結果----------------------------------------20
3.1. 檳榔子萃取物與檳榔素會抑制CHO-K1細胞的生長與造成細胞毒性反應--------------------------------------------20
3.1.1. 短期細胞存活率分析~錐蟲藍排除法---------------------------20
3.1.2. 長期細胞存活率分析~細胞群落形成效率分析-------------------20
3.2. 檳榔子萃取物與檳榔素會改變CHO-K1細胞之細胞形態------------------20
3.3. 檳榔子萃取物與檳榔素會造成細胞內活性氧含量的上升-----------------21
3.4. 檳榔子萃取物與檳榔素會誘導微核的生成-----------------------------21
3.5. 檳榔子萃取物與檳榔素對細胞週期的影響-----------------------------23
3.6. 檳榔子萃取物與檳榔素對細胞死亡的影響-----------------------------23
3.7. 檳榔子萃取物會造成細胞質分裂失敗及雙核細胞之生成-----------------24
3.8. 檳榔子萃取物作用下的雙核細胞仍可維持細胞之分裂-------------------25
3.9. 咖啡因會加成檳榔子萃取物對細胞之毒性反應-------------------------26

第四章 討論-------------------------------------------28
4.1. 檳榔子萃取物與檳榔素對細胞生長的影響-----------------------------28
4.2. 檳榔子萃取物與檳榔素會誘導微核的生成-----------------------------29
4.3. 檳榔子萃取物與檳榔素皆會造成細胞週期的停滯-----------------------30
4.4. 檳榔子萃取物會抑制細胞質分裂之進行，造成雙核，多核，多倍體與非整倍體細胞之增多，且伴隨中心體異常現象之發生-------------------------32
4.5. 咖啡因會加成檳榔子萃取物細胞之毒性反應---------------------------35

第五章 總結----------------------------------------------------------------37

參考文獻--------------------------------------------------------------------38

圖次
Fig. 1A. Particles of the aqueous extract of the areca nut----------------------------------------46
Fig. 1B. Procedures for the preparation of the aqueous areca nut extract ---------------------46
Fig. 2A. Effects of ANE and arecoline to CHO-K1 cells on trypan blue dye exclusion test
-----------------------------------------------------------------------------------------------47
Fig. 2B. Cytotoxicity of ANE and arecoline to CHO-K1 cells determined by clonogenic
cell survival assay-------------------------------------------------------------------------48
Fig. 3A. Morphological alterations of CHO-K1 cells following 24h exposure to ANE-----49
Fig. 3B. Morphological alterations of CHO-K1 cells following 24h exposure to arecoline
------------------------------------------------------------------------------------------------50
Fig. 4. Effects of ANE and arecoline on the cellular production of H2O2--------------------51
Fig. 5A. & B. Effects of ANE and arecoline on the micronuclei frequency of CHO-K1 cells
in the CBMN assay-----------------------------------------------------------------------52
Fig. 5C. The morphology of the micronuclei in the binucleated cells-------------------------53
Fig. 5D. Cytokinesis-block proliferation index (CBPI) of ANE/ arecoline-treated
CHO- K1 cells------------------------------------------------------------------------------54
Fig. 5E. Effects of catalase on ANE- or arecoline-induced micronuclei frequency in
binucleated cells---------------------------------------------------------------------------55
Fig. 6A. Effects of ANE on the cell cycle progression of CHO-K1 cells---------------------56
Fig. 6B. Effects of arecoline on the cell cycle progression of CHO-K1 cells----------------57
Fig. 7A. Morphological alterations of CHO-K1 cells following prolonged exposure to
ANE or arecoline--------------------------------------------------------------------------58
Fig. 7B. Effects of ANE on the cell cycle progression of CHO-K1 cells following different
time exposure------------------------------------------------------------------------------59
Fig. 7C. Effects of arecoline on the cell cycle progression of CHO-K1 cells following
different time exposure-------------------------------------------------------------------60
Fig. 8A. Binucleated and multinucleated cells induced by ANE-------------------------------61
Fig. 8B. Photographs of ANE-treated cells by fluorescence staining with rhodaminephalloidin
and DAPI----------------------------------------------------------------------62
Fig. 8C & 8D. The percentage of binucleated cells following 24h exposure to ANE and
arecoline------------------------------------------------------------------------------------63
Fig. 8E. Preventive effects of catalase on the formation of the binucleated cells following
24h exposure of ANE---------------------------------------------------------------------64
Fig. 9. The percentage of hyperploid cells following exposure to ANE and arecoline
-----------------------------------------------------------------------------------------------65
Fig. 10. Photographs of CHO-K1 cells after 24 h treatment with ANE followed by
fluorescence labeling of γ-tubulin with TRITC and of nuclear DNA with DAPI
-----------------------------------------------------------------------------------------------66
Fig. 11A. Morphological alterations of CHO-K1 cells following prolonged exposure to
ANE, caffeine, and the combination of ANE and caffeine -------------------------67
Fig. 11B. Cytotoxicity of ANE enhanced by caffeine---------------------------------------------68
Fig. 11C. Effects of caffeine on ANE-induced cell cycle alterations and cell death in CHOK1
cells-------------------------------------------------------------------------------------69
Fig. 11D. Morphological alterations of CHO-K1 cells following prolonged exposure to
arecoline, caffeine, and the combination of arecoline and caffeine----------------70
Fig. 11E. Effects of caffeine on the cytotoxicity of arecoline to CHO-K1 cells--------------71
Fig. 11F. Effects of caffeine on arecoline-induced cell cycle alterations and cell death-----72

附錄
附錄-1----------------------------------------------------------------------73
附錄-2----------------------------------------------------------------------74

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