系統識別號 U0026-0802201217213100
論文名稱(中文) 人類鹼基切除修復因子 nth endonuclease III-like 1 之功能性探討
論文名稱(英文) Functional characterization of the human base excision repair factor nth endonuclease III-like 1
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系碩博士班
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 100
學期 1
出版年 101
研究生(中文) 李芸萍
研究生(英文) Yun-Ping Lee
學號 t36981087
學位類別 碩士
語文別 中文
論文頁數 51頁
口試委員 口試委員-謝淑珠
中文關鍵字 鹼基切除修復 
英文關鍵字 base excision repair  nthl1  ogg1 
中文摘要 從環境因子及內在代謝所產生的氧化性壓力是造成DNA修飾的主要來源, DNA修飾會造成突變及基因組不穩定的現象。在哺乳類動物細胞中,主要由兩個DNA醣苷酶, nth endonuclease III-like 1 (NTHL1) 及 8-oxoguanine DNA glycosylase 1 (OGG1), 經由鹼基缺除修復系統 (base excision repair, BER) 來移除氧化性的DNA損傷。 先前的研究已經發現OGG1在DNA修復及細胞生存能力的維持扮演重要的角色,但是NTHL1在氧化性壓力所造成的DNA損傷及修復所扮演的角色尚未很仔細的被探究。為了闡明NTHL1在氧化性壓力所造成的DNA損傷修復所扮演的角色,以及了解NTHL1及OGG1彼此間在DNA修復中是否有協同性作用,我們利用以慢病毒為載體的shRNA送入系統 (lentivirus-based shRNA system) 建立了 nthl1及ogg1 穩定knock-down的細胞株。利用MTT assays我們發現nthl1及ogg1 knock-down之細胞對氧化壓力較為敏感,表示NTHL1對於修復氧化性壓力造成的DNA損傷是必要的。而且,由Annexin V-FITC 凋亡分析實驗及以及細胞週期之分析發現,所有的knock down細胞在氧化性壓力下,細胞凋亡的比例會大為增加。此結果也指出NTHL1 對於維持DNA損傷後細胞的生存能力也是必須的。雖然之前的研究顯示NTHL1會參與在8oxoG的移除,但是,我們利用活體外鹼基切除修復實驗 (in vitro BER assay) 得到的結果顯示NTHL1參與在thymine glycol的移除,並不參與8ooxG的切除。除此之外,利用RT-PCR,我們知道nthl1及ogg1 不不會因為氧化壓力而影響其mRNA之表現。而nthl1 及 ogg1 knock-down 不會改變nei endonuclease VIII-like 1/2的表現。總結來說,NTHL1 在細胞遭受氧化性壓力下對於維持細胞之生存扮演重要的角色,並且參與thymine glycol的切除,而非8oxoG。
英文摘要 Oxidative stress from environmental agents and endogenous metabolic processes is a common source to cause DNA modifications, which result in mutations and genomic instability. In mammalian cells, two major DNA glycosylases nth endonuclease III-like 1 (NTHL1) and 8-oxoguanine DNA glycosylase 1 (OGG1) remove oxidative DNA damage through the base excision repair (BER) pathway. Previous studies have found that OGG1 plays an important role in DNA repair and maintaining cell viability. But the role of NTHL1 in oxidative stress-induced DNA damage and repair was not extensively examined. To elucidate the role of NTHL1 in oxidative stress-induced DNA repair and to know whether NTHL1 and OGG1 have synergistic effects with each other in DNA repair, we established stable nthl1, ogg1, and nthl1/ogg1 double knock-down (KD) cells by using lentivirus-based shRNA system. Using MTT assays, we found that all the KD cells examined were sensitive to oxidative stress, suggesting that NTHL1 is required for repairing oxidative DNA lesions. Also, all of the KD cells exhibited increased apoptosis under oxidative stress, shown by Annexin V-FITC apoptosis assays and cell cycle analysis, which indicates that NTHL1 is also required for maintaining cell viability after DNA damage. Although previous studies indicated NTHL1 can remove 8oxoG, by the in vitro BER assay, we investigated that NTHL1 involved in thymine glycol removal, but not 8oxoG. By RT-PCR, we found nthl1 and ogg1 were not stress-inducible, and nthl1 and ogg1 knock down didn’t change the expression of nei endonuclease VIII-like 1/2. In summary, NTHL1 plays important role in maintaining cell viability under oxidative stress and involves in DNA damages removal of thymine glycol, not 8oxoG.  
論文目次 中文摘要 I
Abstract II
目錄 III
表目錄 V
圖目錄 VI
附錄目錄 VII
第一章 緒論 1
1.1 DNA損傷及其修復 1
1.2 氧化性壓力、氧化性DNA損傷及其相關疾病 3
1.3 鹼基切除修復(Base excision repair, BER) 5
1.4 nth endonuclease III-like 1 (NTHL1) 6
1.5 8-oxoguanine DNA glycosylase 1(OGG1) 7
第二章 研究動機 9
2.1 探討nthl1 及ogg1 knock down細胞株之表型特徵 9
2.2 探討NTHL1對DNA損傷之作用物 10
第三章 實驗材料與方法 11
3.1細胞株與化學藥品 11
3.2建立nthl1及ogg1穩定 knock down之細胞株 11
3.2.1病毒製造 11
3.2.2病毒感染 11
3.2.3萃取核糖核酸(RNA) 12
3.2.4反轉錄聚合酶連鎖反應(RT-PCR) 12
3.2.5西方墨點法 (Western blot) 13
3.3細胞存活率試驗 (MTT assay) 14
3.4凋亡細胞分析 15
3.5細胞週期分析 15
3.6活體外鹼基切除修復分析(In vitro BER assay) 16
3.6.1萃取細胞蛋白(Whole cell extract) 16
3.6.2活體外鹼基切除修復 16
3.7無鹼基位點(abasic site)偵測分析 17
3.7.1萃取細胞之DNA 17
3.7.2 西南方墨點法(south-western blotting) 18
第四章 結果 19
4.1 建立穩定knock down nthl1及ogg1之細胞株 19
4.2 在氧化壓力下nthl1及ogg1 knock down細胞株的生存能力 19
4.3 氧化性DNA損傷會導致nthl1及ogg1 knock down細胞株的凋亡 20
4.4 氧化性DNA損傷會導致nthl1及ogg1 knock down細胞株細胞週期停滯,進而導致細胞的死亡 21
4.5 nthl1及ogg1 mRNA表現不會因為menadione及UV所造成的傷害而改變 22
4.6 NTHL1參與thymine glycol的移除,並不參與8-oxoG的移除 22
4.7 NTHL1及OGG1的缺失不影響neil 1/2的表現 23
第五章 討論 24
第六章 Reference 28
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