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研究生:宋政達
研究生(外文):Jheng Da Song
論文名稱:幽門螺旋菌抑制宿主細胞修復氧化壓力造成的DNA損傷但不增加突變率
論文名稱(外文):Helicobacter pylori Inhibits Repair of DNA Damage Caused by Oxidative Stress but Does Not Increase Mutation Rate in Host Cells
指導教授:邱全芊
指導教授(外文):C. C. Chiou
學位類別:碩士
校院名稱:長庚大學
系所名稱:醫學生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
論文頁數:58
中文關鍵詞:幽門螺旋菌DNA修復彗星試驗突變率試驗
外文關鍵詞:Helicobacter pyloriDNA repairBleomycinMNNGComet assayhprt mutation assay
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胃癌是目前癌症死因中排名第二名的癌症,在台灣地區則為第五名。胃癌可由多個因子影響造成,其中最有可能的便是幽門螺旋菌。幽門螺旋菌是屬於格蘭氏染色陰性菌,可寄生於人類胃部中,其致癌機制可能是位於胃部產生大量活性氧分子(ROS)。本研究的假說是幽門螺旋菌不只產生ROS,也會抑制胃細胞修復ROS所造成的傷害。為了檢驗此假說,我們將細胞與幽門螺旋菌共培養後,再使用Bleomycin (BLM)及MNNG引發DNA損傷。在彗星試驗中,發現了受到幽門螺旋菌感染的細胞,修復DNA損傷的能力顯著地降低了。但是藉由分析hprt基因的突變率時,BLM及MNNG造成的突變率卻無增加的現象。於本研究中發現,幽門螺旋菌會抑制宿主細胞修復氧化壓力所造成的DNA損傷,但是不增加突變率。
Gastric cancer is the world’s second commonest malignancy. The most potential gastric carcinogen responsible for gastric cancer is the infection of Helicobacter pylori, a gram-negative bacterium resident in gastric mucosa. One of the possible mechanisms that H. pylori causes cancer is that it generates large amount of reactive oxygen species (ROS) in gastric mucosa. Our hypothesis is that H. pylori not only produce ROS, but also inhibit repair of the ROS-caused DNA damage. To test this hypothesis, we investigated the repair ability in gastric cells for Bleomycin (BLM)- and MNNG-caused DNA damage after coincubation with H. pylori. Measuring the tail moment with comet assay, we found that the repair of strand breaks induced by BLM significantly decreased in H. pylori-infected cells. Analyzing the hprt mutation rate with 6-thioguanine assay, we found that H. pylori infection does not increase BLM- and MNNG-induced mutation rate. In conclusion, H. pylori may inhibit the repair ability and enhance ROS-induced DNA damage but does not increase mutation in gastric cells.
指導教授推薦書 ..............................i
口試委員審定書 ............................ii
誌謝 ..................................iii
摘要......................................iv
Abstract ..................................v
英文名詞縮寫對照表 ..........................vi
目錄......................................vii
圖表目錄....................................ix
第一章 緒論 ....................................1
1.1 胃癌的流行病學..............................1
1.2 影響胃癌發生的因子 ......................... 2
1.3 飲食與胃癌 .............................. 2
1.4 幽門螺旋菌與胃癌的關係.......................3
1.5 活性氧分子與胃癌的關係.......................4
第二章 實驗目的與設計............................6
2.1 假說與目的 .................................6
2.2 實驗設計....................................6
第三章 材料與方法................................8
3.1 藥品及試劑 .................................8
3.2 細胞株及其培養 ..............................9
3.3 菌株及其培養 ................................9
3.4 細胞與幽門螺旋菌的共培養......................11
3.5 BLM處理細胞之方式 ...........................11
3.6 MNNG處理細胞之方式...........................12
3.7 彗星試驗....................................12
3.8 細胞群落形成分析
(colony forming assay).....................14
3.9 突變率分析...................................14
3.10結晶紫染劑的配製、染色及計數原則 ...............15
第四章 結果......................................16
4.1 H.pylori與BLM對細胞的共同作用 .................16
4.1.1 不同菌株對AGS細胞的DNA修復能力的影響...........16
4.1.2 不同菌株對TSGH細胞DNA修復能力的影響............17
4.1.3 NCTC-11637對TSGH細胞修復8-oxoG的影響.........19
4.1.4 NCTC-11637菌株對TSGH細胞突變率的影響 .........20
4.2 NCTC-11637菌株對MNNG所造成的細胞突變率的影響.....21
第五章 討論........................................23
參考文獻...........................................25

表一、各H. pylori 菌株基因及生化特性.................33
圖一、BLM處理1小時TSGH之存活率.......................34
圖二、彗星試驗中BLM濃度的選擇........................35
圖三、NCTC-11637感染AGS細胞後,細胞修復DNA的能力......36
圖四、BCRC-17026感染AGS細胞,細胞修復DNA的能力 .........37
圖五、TSGH細胞感染不同H. pylori菌株及處理BLM後所進行的彗星試驗.....................................................38
圖六、不同H. pylori菌株對TSGH細胞的DNA修復能力的影響.......40
圖七、Fpg-modified彗星試驗定量圖.........................41
圖八、NCTC-11637對BLM所造成的TSGH細胞突變率的影響..........42
圖九、MNNG處理TSGH細胞的存活率及突變率.....................43
圖十、H. pylori對於MNNG造成的細胞突變率的影響...............44
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