張美姬臺灣大學:口腔生物科學研究所王維霆Wang, Wei-TingWei-TingWang2010-05-042018-07-092010-05-042018-07-092009U0001-3007200916131900http://ntur.lib.ntu.edu.tw//handle/246246/178625嚼食檳榔是一項普遍風行於亞洲、非洲、歐洲部分地區各個社會階層的流行嗜好，尤其是泰國、馬來西亞、印尼、印度、中國、台灣等地區，根據統計，全世界有嚼食檳榔習慣的人口達到六億人之譜。嚼食檳榔也被證明是多種口腔粘膜病變：口腔白斑、口腔黏膜下纖維化甚至口腔癌的主要致病因子之一，國際癌症研究中心也發表專論指出，檳榔子本身即為一級致癌物，許多研究指出檳榔萃取物的確存在著具細胞與基因毒性物質，能夠造成DNA斷裂、DNA與蛋白質交互連結、非正常程序的DNA合成、細胞週期改變等異常現象。目前已經有研究指出，當細胞發生DNA傷害時，會有ATM/ATR-Chk1/Chk2路徑的活化，使細胞週期停滯，並活化其他參與DNA修補蛋白的表現。但由於檳榔成分過於複雜，對於檳榔成分究竟是如何造成各種細胞變異甚至口腔疾病的詳細致病機轉目前並不清楚，因此本篇研究的主要目的即是針對細胞內兩種細胞週期檢查點Chk1/Chk2在檳榔成分所造成的基因與細胞毒性中所扮演的角色進行探討。在dose dependent的實驗中發現，隨著檳榔子萃取物與檳榔素濃度的提高，SAS舌癌細胞不只外型出現了明顯變圓，較不貼附於培養皿的現象，細胞內也出現大量空泡。在細胞的存活性實驗也發現，隨著檳榔子萃取物與檳榔素濃度提高，分別在800 ug/ml與0.8 mM濃度時，SAS細胞存活率會有明顯下降的情況。利用流式細胞儀進行細胞週期分析也發現隨著檳榔子萃取物濃度提高，SAS細胞會有G2/M phase arrest的現象。進一步使用RT-PCR與西方點墨法研究檳榔子萃取物與檳榔素是否能有效活化各個與細胞週期調控以及細胞凋亡有關之指標分子表現，發現檳榔子萃取物與檳榔素能夠活化細胞內Chk2訊息傳遞路徑表現，藉由Chk2的磷酸化，進一步促進下游分子如p-Cdc25C、p-Cdc2表現，而Chk2的活化同時也影響了與細胞週期有關的cyclin B1、cyclin D表現，檳榔子萃取物與檳榔素也能調控和細胞凋亡有關的p-p53、Bcl-2、Bax在細胞內的表現。而ATM/ATR抑制劑咖啡因與Chk2抑制劑的使用則更進一步的釐清了Chk2訊息傳遞路徑中各分子的上下游關係，令人意外的是，使用Chk2抑制劑降低Chk2表現後，SAS細胞的G2/M phase arrest現象消失了，取而代之的是細胞大量堆積在G1時期。總結來說，我們提供了檳榔子萃取物與檳榔素對於細胞變異的可能分子機制，檳榔子萃取物與檳榔素能夠活化SAS細胞Chk2訊息傳遞路徑，證實了Chk2的表現在檳榔成分引起的細胞變異中的確扮演著舉足輕重的角色，而在未來臨床應用上，Chk2也許可以做為檳榔引起的口腔疾病治療中一個新的標的。Betel quid (BQ) chewing is a very common habit in Asia, Africa, and a portion of Europe. It enjoys complete social acceptance in many societies especially in Thailand, Malaysia, Indonesia, India, China and Taiwan. It has been estimated that there are about 6 million BQ-chewers living in different regions of the world. BQ chewing is demonstrated to be one of the major risk factors leading to leukoplakia, oral submucous fibrosis, and oral cancer. In a monograph published by the International Agency for Research on Cancer（IARC）for the evaluation of cancer risks (IARC, 2004), areca nut was ranked as a group I carcinogen to humans. There are many reports indicating the components of areca nuts have genotoxicity and cytotoxicity leading to DNA strands breaks, DNA-proteins crosslink, unschedualed DNA synthesis and cell cycle aberration. When DNA damage occurs, the ATM/ATR-Chk1/Chk2 pathway is activated, cell cycle arrested, and induces the activation of other DNA-repair proteins. So far, because of the components of AN are very complex, the mechanisms of AN-induced cytotoxicity to cause cell aberrations and oral diseases are not clearly understood. The purpose of this study is trying to investigate the roles of two check point kineses: Chk1/Chk2 in the ANE-induced geno- and cytotoxicity. In the ANE-dose dependent experiments, the morphology of SAS cells became much roundly, lost the connection to the plate and many vacuoles appeared in the cells due to the increased ANE-concentration. The viability of SAS cells decreased obviously because of the raised of the ANE and arecoline, especially in the concentration of 800 ug/ml and 0.8 mM respectively. By using flow-cytometry assay, we found that the SAS cells arrested in G2/M phase because of the effects of the components of areca nuts. We further analysed whether the ANE and the arecoline can regulate the cell cycle- and apoptosis-related molecules by RT-PCR and western blot. ANE and arecoline activated the Chk2 pathway and induced the downstream p-Cdc2, p-Cdc25C expression through the Chk2 phosphorylation. The ANE and arecoline also regulated the cell cycle-related cyclin B1, cyclin D1 and the apoptosis-related p-p53, Bax and bcl-2. By using caffeine, an ATM/ATR inhibitor and Chk2 inhibitor, we clarified the correlation between the molecules of the ATM-Chk2 signaling transduction pathway. Unexpectedly, Chk2 inhibition not only blocked the ANE-induced G2/M phase arrest of SAS cells but also resulted in the G1 phase arrest. In conclusion, we provide the possible molecular mechanism of Chk2 pathway, which is induced by ANE and arecoline to cause aberration in SAS cells. Chk2 activation indeed plays a very important role in the cell mutagenesis induced by areca nuts components. Maybe Chk2 can be the new target in the prevention and clinical treatment of the areca nuts-induced oral mucosa diseases in the future.第ㄧ章　緒論 1榔嚼食與口腔疾病 1榔嚼塊之組成成分 1榔萃取物的基因毒性、細胞毒性與致癌性 2胞週期 3yclin B 4DK1-Cdc2 5dc25C 6查點（Check Points） 6CL2 Family與細胞週期 8究動機與目的 8二章　實驗材料與方法 10. 檳榔子成分萃取 10. 細胞株與細胞培養 10. 細胞生長評估 11. MTT（3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide）分析 12. 流式細胞儀（Flow Cytometry）分析 13. 西方點墨法（Western Blotting） 14.1 細胞處理與蛋白質萃取 14.2 蛋白質定量 14.3 膠體配製與電泳 15.4 蛋白質轉漬（Transfer） 16.5 抗體使用 16. 聚合酶連鎖反應（polymerase chain reaction） 18.1 RNA分離法 18.2 RNA定量 19.3 RNA反轉錄（Reverse Transcription） 20.4 聚合酶連鎖反應（Polymerase Chain Reaction, PCR） 20. 統計分析 22三章　實驗結果 23榔成分對於SAS舌癌細胞之影響 23榔成分與SAS舌癌細胞型態變異 23榔子萃取物導致SAS舌癌細胞型態變異 23榔素（Arecoline, ACO）導致SAS舌癌細胞型態變異 24榔成分之細胞毒性 24榔子萃取物與細胞毒性 25榔素與細胞毒性 25榔成分對SAS舌癌細胞之細胞週期的影響 26榔子萃取物對SAS細胞之細胞週期的影響 26榔素對SAS細胞之細胞週期的影響 27榔成分影響SAS細胞之細胞週期調控分子 27榔子萃取物對SAS舌癌細胞之細胞週期調控分子的影響 28榔素對SAS舌癌細胞之細胞週期調控分子的影響 29TM/ATR在檳榔子萃取物對SAS舌癌細胞所造成之影響中所扮演之角色 30啡因對檳榔子萃取物引起的SAS舌癌細胞外表型態變異之影響 30hk2在檳榔子萃取物對SAS舌癌細胞所造成之影響中所扮演之角色 31制Chk2對檳榔子萃取物引起的SAS細胞外表形態變異之影響 31制Chk2對檳榔子萃取物細胞毒性之影響 32制Chk2對檳榔子萃取物引起的SAS細胞之細胞週期變異之影響 32制Chk2影響檳榔子萃取物調控SAS細胞之細胞週期分子 33四章　討論 35榔子萃取物與檳榔素對於細胞生長的影響 35榔成分活化Check Point Kinase 2（Chk2） 36TM與Chk2對SAS細胞存活能力的影響 37hk2對SAS細胞之細胞週期調控分子的影響 38五章　總結 41六章　參考文獻 43次： 52igure 1：Effect of AN extraction on morphological changes in SAS cells. 52igure 2：Effect of Arecoline on morphological changes in SAS cells. 53igure 3：Dose dependent effects of ANE on cells proliferation in SAS cells. 54igure 4：Dose-dependent effects of ANE on SAS cells. 55igure 5：Dose-dependent effects of ACO on SAS cells. 56igure 6：Effect of ANE on the expression of p-Chk2、Chk2、p-Chk1 and Chk1 in SAS cells. 57igure 7：Effect of ANE to expression of cell cycle regulatory proteins in SAS cells. 58igure 8：Effect of ANE to mRNA expression of cell cycle regulatory proteins in SAS cells. 59igure 9：Effect of ACO on the expression of p-Chk2、Chk2、p-Chk1 and Chk1 in SAS cells. 60igure 10：Effect of ACO to expression of cell cycle regulatory proteins in SAS cells. 61igure 11：Effect of ACO to mRNA expression of cell cycle regulatory proteins in SAS cells. 61igure 12：Effect of caffeine on ANE-induced morphological changes in SAS cells. 63igure 13：Effect of Chk2 inhibitor on ANE-induced morphological changes in SAS cells. 64igure 14：Dose-dependent effects of ANE - Chk2 inhibitor on cells proliferation in SAS cells. 65igure 15：Dose-dependent effects of ANE - Chk2 inhibitor on SAS cells. 66igure 16：Effect of ANE-Chk2 inhibitor to expression of p-Chk2 and Chk2. 67igure 17：Effect of Chk2 inhibitor on ANE-induced changes of cell cycle-related proteins. 68igure 18：Schematic diagram illustrating possible molecular mechanism of areca nuts components-induced Chk2 activation. 69錄： 70錄一　中華民國96年度十大癌症死因 70錄二　The cell cycle 71錄三　Multiple CDKs & cyclins regulate passage of the cell cycle 71錄四　The effect of Cdc25 on cyclin/CDK 71錄五　The check point signaling 72錄六　The mechanism of apoptosis 73application/pdf3726631 bytesapplication/pdfen-US檳榔子萃取物檳榔素Chk2細胞毒性細胞週期ANEarecolinecytotoxicitycell cycle[SDGs]SDG3http://ntur.lib.ntu.edu.tw/bitstream/246246/178625/1/ntu-98-R96450016-1.pdf