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研究生:鄭亮森
論文名稱:檳榔嚼塊各成分誘導DNA斷裂和DNA—proteinCross-link(DPC)的能力
指導教授:郭英雄郭英雄引用關係韓良俊韓良俊引用關係鄭景暉鄭景暉引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:臨床牙醫學研究所
學門:醫藥衛生學門
學類:牙醫學類
論文種類:學術論文
論文出版年:2000
畢業學年度:88
語文別:中文
論文頁數:62
中文關鍵詞:檳榔子荖花石灰檳榔嚼塊DNA斷裂自由基0.7%電泳膠片檳榔素
外文關鍵詞:DNA-protein Cross-linkDNA breakPUC 18 DNApBR 322 DNAFenton reactiondesferrioxamineAreca nutInflorescence Piper Betle
相關次數:
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檳榔嚼塊各成分誘導DNA斷裂 和DNA — protein Cross- link (DPC) 的能力
總摘要
因為臺灣相當高的口腔癌罹患率被認為與國人嚼食檳榔嚼塊的習慣有密切的關連,因此檳榔嚼塊中各種成分在誘導口腔癌形成所扮演的角色值得進一步去評估。在第一章節中,我們測試檳榔嚼塊各成分誘發pBR322 DNA的破壞狀況,在檳榔子萃取液、荖花萃取液、CuCl2溶液、Ca(OH) 2溶液、檳榔子萃取液加CuCl2溶液、荖花萃取液加CuCl2溶液和荖花萃取液加H2O2各種反應下,只可見pBR322 DNA單股斷裂。pBR322 DNA雙股斷裂則見於FeCl2溶液,檳榔子萃取液加FeCl2溶液和荖花萃取液加FeCl2溶液三個反應中。而FeCl2溶液加H2O2和CuCl2溶液加H2O2的反應則造成pBR322 DNA嚴重破壞甚至消失。一種非常奇特的現象可見於檳榔子萃取液和H2O2的反應中,沉澱物快速出現,若加入DNA 則DNA單股斷裂、模糊層和DNA 停滯於膠片頂端原位同時出現。DNA滯留在膠片頂端原位的情況在加入desferrioxamine(一種鐵離子箝合劑)後可部分被阻斷。因此推論此沉澱物之產生與鐵有關。這種DNA 停滯不前的現象可被解釋成DNA-protein 連結或DNA-DNA連結。
化學物質誘發DNA-DNA、DNA-protein cross-links之特性已被發現與cancer之誘導有密切關係,因此我們感到非常興趣的是:是否檳榔嚼塊內各成分或成分間相互作用在體外試驗是否亦能誘導DDC或DPC的形成。在第二章節中,我們採用PUC18 DNA和BSA(bovine serum albumin)與檳榔嚼塊內各種成分在50mM pH 7.0 Tris-HCl buffer solution下進行反應。在實驗中,我們證實檳榔子萃取液和H2O2作用也可產生類似DDC或DPC的現象,但是若檳榔嚼塊內各成分單獨和BSA作用,則無cross-links的現象出現。
由於DNA受損和突變的形成有密切的關係。而基因突變更在癌的形成過程中扮演重要的角色。而檳榔嚼塊內存在有造成DNA斷裂和保護DNA免於斷裂的成分可能是檳榔口腔癌的形成為何須要長時間的原因(10年20年或更久)。
Abstract
The high incidence of oral cancer in Taiwan is related to betel quid (BQ) chewing. However the roles of various BQ ingredients in inducing oral cancer formation deserve further evaluation. In the first chapter, we test whether chemical reactions of BQ ingredient can induce strand breaks of pBR322 DNA. Only single strand break was noted when pBR 322 plasmid DNA was incubated with the areca nut(AN) extract , piper betle inflorescence(IPB)extract , CuCl2、Ca(OH) 2、AN extract with CuCl2 , IPB extract with CuCl2 , and AN extract with H2O2 . Double strand breaks was noted when pBR 322 DNA was exposed to FeCl2, AN extract with FeCl2 and IPB extract with FeCl2。pBR 322 DNA is completely degraded (or fragmented) when exposed to FeCl2 with H2O2, or CuCl2 with H2O2. Interestingly, precipitation was seen immediately after incubation of DNA with AN extract and H2O2. Single strand break, smear layer and DNA left on the top DNA were observed at the group. This phenomenon may be explained by the occurrence of DNA-protein cross-link or DNA-DNA cross-link. DNA left at top gel well can be blocked by the addition of desferrioxamine (a iron chelating agent). The precipitant should contain iron.
DNA-DNA cross-links (DDC) or DNA-protein cross-links are difficult for repair and show strong correlation to the carcinogenic potential of chemicals. It is therefore very interesting to realize whether the ingredients of betel quid can induce DDC or DPC formation. In the second chapter, we tested the interactions among PUC 18 DNA, bovine serum albumin (BSA) and the individual ingredients of betel quids at 50mM Tris-HCl buffer solution, pH 7.0. The result of DNA, AN extract and H2O2 reaction is also similar to DDC or DPC formation. Direct incubation of betel quid ingredients with BSA reveal no cross-link formation.
There is a very close relationship between DNA damage and mutation. Mutation of gene plays a very important role in the carcinogenesis. The coexisting property of DNA-nicks and DNA protection in BQ may be the reason why the formation of oral cancer need a very long time (ten, twenty years or more)。
目錄
總摘要 頁數
中文摘要……………………………………………………………1
英文摘要…………………………………………………………….3
Part I:檳榔嚼塊內各成分誘導DNA斷裂的能力
一、前言 ……………….………………………………..….….5
二、註解 ……….……………………………………………….7
註1檳榔嚼塊的成分
   註2檳榔子所含的成分
   註3Fenton reaction
   註4Catalase and peroxidases
   註5desferrioxamine
三、中文摘要… ……………………………………………… 12
四、英文摘要 ………………………...……………………….13
五、文獻回顧
第一節嚼食檳榔造成口腔黏膜變化的證據……………………..……15
第二節檳榔嚼塊成分和DNA的作用模式……………………..…….16
第三節自由基與口腔黏膜疾病………………………………….…….21
第四節DNA斷裂和癌形成的關連……………………………….…...22
六、研究目的…………………………………………………..24
七、材料與方法……………………………………..……..…..25
八、結果……………………………………………….…….... 28
九、討論……………………………………………………………30
十、結論……………………………………………………………39
Part II:檳榔嚼塊內各成分誘導DNA-protein Cross-link (DPC)的能力
一、中文摘要………………………………………………….…..41
二、英文摘要……………………………………………………....42
三、文獻回顧
第一節 DNA-protein Cross-link (DPC)……………………………….……...43
第二節 檳榔萃取物誘導DNA-protein Cross-link (DPC)形成的可能機轉..44
第三節 DNA-protein Cross-link (DPC)與Cancer之關係…………………..45
四、研究目的……………………………………………………...47
五、材料與方法……………………………………………………48
六、結果……………………………………………………………52
七、討論……………………………………………………………54
八、結論……………………………………………………………60
九、附錄……………………………………………………………62
附圖
附表
參考文獻
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