博碩士論文 102224015 詳細資訊




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姓名 邱炳豪(Bin-Hao Chiou)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 4-aminobiphenyl誘導HepG2細胞中的microRNAs表現 並藉由microRNAs調控DNA修復機制
(MicroRNA regulation of DNA repair gene expression in4-aminobiphenyl-treated HepG2 cells)
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★ Pseudomonas sp. A46全基因組分析與重金屬復育基因工程菌開發★ 4-Aminobiphenyl 調控 miR-630 抑制 RAD18 表現誘導 Hep3B 細胞產生氧化性 DNA 損傷
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摘要(中) 流行病學研究指出,當生物體長期暴露在4-胺基聯苯 (4-aminobiphenyl,4-ABP) 的環境下,會造成肝臟細胞的DNA damage與DNA adducts,甚至會誘導膀胱癌的形成。4-ABP進入到人體後,會藉由肝臟細胞的phase I與phase II的氧化還原進行代謝,但過程中會產生自由基,進一步地導致DNA 雙股斷裂。本篇研究主要深入探討,4-ABP所造成的DNA損傷,其細胞內的自體修復機制為什麼不能立即做修補,是不是還有其他因素來調控修復機制? 首先利用comet assay來觀察DNA damage程度,處理4-ABP (0-300M)後,發現4-ABP確實會造成HepG2 cell的DNA損傷。PCR array與miRNAs microarray結果亦顯示,有16個關於DNA repair相關的基因表現下降,27個miRNAs 表現差異高達三倍以上,其中miR-630與miR-513a-5p為最顯著的表現。因此想深入探討,其DNA repair相關基因的失調,是否因為miRNAs所調控。研究結果顯示,miR-630會target在RAD18與EXO1的3’UTR上、miR-513a-5p則會target在XRCC2與FANCG之3’UTR。利用質體轉染技術,讓細胞內的miR-630和miR-513a-5p大量表現或抑制,皆會影響RAD18、XRCC2與FANCG的蛋白質表現。有文獻指出,4-ABP最主要會造成HepG2 cell的氧化性基因損傷並導致DNA double strands break,因此以miR-513a-5p與XRCC2為本研究主軸。
4-ABP (75M) 與自由基抑制劑NAC結合處理,發現4-ABP誘導ROS的產生,會使miR-513a-5p大量表現,並進一步地導致XRCC2蛋白表現失調。將XRCC2 蛋白大量表現,其DNA damage的程度也有顯著性的被修復。本研究證明了4-ABP、oxidative stress、miR-513a-5p、XRCC2與DNA damage之間的互相調控機制,並釐清4-ABP所造成的DNA damage,其DNA repair system為什麼不能立即修復。最後,本研究提供了一個4-ABP所造成肝臟細胞的氧化基因損傷之預防檢測與治療的方向。
摘要(英) The study goal was to evaluate the effects of 4-aminobiphenyl (4-ABP) on DNA damage based on the regulation of miRNAs to suppress some reducing DNA-repair proteins in human HepG2 cells. In this study, we used comet assay to determine that 4-ABP (0–300 M) induces DNA damage in HepG2 cells after 24 hours. DNA damage signaling pathway-based PCR arrays were used to investigate differential expressed genes response to 4-ABP treatment. In parallel, the miRNAs array was revealed that the expression of 27 miRNAs in 4-ABP-treated cells was at least 3-fold higher than that in the control group. Of these 27 miRNAs, the most significant expression of miRNA-513a-5p and miRNA-630 was further validated by qRT-PCR, which was predicted to target to RAD18、XRCC2 and FANCG genes, respectively, via bioinformatic analysis. In addition, overexpression and knockdown of miRNA-630 and miRNA-513a-5p inversely regulate the expression of RAD18、XRCC2 and FANCG proteins expression levels. 
We found that ROS production is crucial for 4-ABP-induced miR-513a-5p expression and this phenomenon would be abolished by ROS inhibitor N-acetylcysteine. XRCC2 overexpression that the extent of DNA damage will be eminently repaired as well. Based on these, we indicated the mechanism of 4-ABP→ROS→miR-513a-5p --| XRCC2→DNA damage, and provide a potential application for prevention and therapy in future.
關鍵字(中) ★ 4-胺基聯苯
★ 氧化壓力
★ miR-513a-5p
★ XRCC2
★ DNA損傷
關鍵字(英) ★ 4-aminobiphenyl
★ oxidative stress
★ miR-513a-5p
★ XRCC2
★ DNA damage
論文目次 目錄
致謝 IV
中文摘要 V
英文摘要 VI
第壹章、緒論 1
第貳章、實驗目的和實驗架構 8
第參章、材料與方法 9
第一節、實驗材料 9
第二節、實驗方法 16
一、細胞株來源、解凍及繼代培養 16
二、細胞計數 17
三、細胞存活率分析 18
四、細胞內活性氧化物質之測定 19
五、測定DNA損傷程度之彗星試驗 20
六、抽取細胞之total RNA 22
七、反轉錄作用 23
八、DNA/RNA電泳 24
九、蛋白質萃取 25
十、蛋白質濃度測定 26
十一、蛋白質電泳 27
十二、蛋白質轉漬 28
十三、西方免疫墨點法 29
十四、即時定量聚合酶連鎖反應 30
十五、大腸桿菌勝任細胞製備 31
十六、大腸桿菌勝任細胞之轉型作用 32
十七、利用限制酵素切開雙股DNA 34
十八、插入子與載體接合 35
十九、酒精沉澱DNA 35
二十、質體抽取 36
二十一、多點突變 37
二十二、質體及RNAi轉染 38
二十三、螢光酶活性測試 39
第肆章、結果 41
第伍章、討論 48
參考文獻 57
圖表 65
附錄 96
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指導教授 陳師慶(Ssu-Ching Chen) 審核日期 2015-7-29
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