博碩士論文 105821028 詳細資訊




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姓名 李佳芸(Chia-Yun Lee)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 探討人類肝癌細胞HepG2經4-氨基聯苯處理過後miRNA-630對於同源重組修復相關蛋白MCM8的調控機制
(Molecular mechanisms of microRNA-630 regulating MCM8 expression in 4-aminobiphenyl-treated HepG2 cells)
相關論文
★ 4-aminobiphenyl誘導HepG2細胞中的microRNAs表現 並藉由microRNAs調控DNA修復機制★ 研究Dicrotophos對HepG2細胞毒性之分子機制:CSA蛋白質在毒性扮演之角色
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★ 4-Aminobiphenyl 調控 miR-630 抑制 RAD18 表現誘導 Hep3B 細胞產生氧化性 DNA 損傷★ 三硝基甲苯之毒理機制及生物降解暨多氯乙烯汙染模場生物整治
★ 假單胞菌Pseudomonas sp. A46之基因工程菌開發及重金屬之生物累積和生物吸附潛力探討
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摘要(中) 4-氨基聯苯已被認定為人類致癌物質,且廣泛存在於日常生活中。先前的研究中證實,4-氨基聯苯會造成ROS的上升及誘導調控DNA修復蛋白的miRNA生成,但對於兩者在4-氨基聯苯對於人類肝癌細胞的毒理機制中扮演的角色仍所知甚少。研究中利用生物資訊軟體TargetScan預測,得知DNA雙股斷裂修復蛋白MCM8會受miRNA-630調控,再藉由觀察-H2AX的表現量偵測DNA雙股斷裂程度。經實驗證實miRNA-630會抑制MCM8的生成,並確實會導致同源重組修復效率下降,抑制DNA雙股斷裂修復。同時也在合併處理4-氨基聯苯與NAC(N-acetyl-1-cysteine)的細胞中,發現miR-630的生成及DNA損傷程度皆有降低,故得知ROS會誘導miRNA-630且參與DNA雙股斷裂修復的調控。
  以上結果證實,經4-氨基聯苯處理後細胞會產生ROS,然後誘導miRNA-630表現,導致DNA修復蛋白MCM8表現量被抑制,最終造成的DNA損傷。
摘要(英) 4-ABP (4-Aminobiphenyl) is recognize as a human carcinogen, and commonly found in our daily life. Previous studies have shown that 4-ABP causes oxidative DNA damage and induces expression of some miRNAs involved in the regulation of DNA repair. However, the mechanism about the role of miRNAs involved in oxidative DNA damage in 4-ABP-treated hepatoma cells is unclear. By using TargetScan software, we found that miR-630 is a conserved target of MCM8, a DNA double-strand breaks repair protein. Therefore, we want to figure out the effects of miRNA on MCM8 expression. Our results proved that the elevated expression of miR-630 resulted in the decrease of MCM8 expression, causing the significant decrease of homologous recombination activity. Accordingly, the repair of DNA double-strand breaks (DSBs) was attenuated as evidenced from γ-H2AX analysis. In 4-ABP-treated cells, we found that the levels of miR-630 expression and DNA damage were attenuated by co-treatment with N-acetyl-1-cysteine (NAC), indicating that ROS-dependent miR-630 was involved in DSBs repair.
  Collectively, 4-ABP induces ROS (Reactive oxygen species) generate, increases miRNA-630 expression, and then attenuates repair of DNA double-strand breaks by targeting MCM8.
關鍵字(中) ★ 4-氨基聯苯
★ miRNA-630
★ MCM8
★ DNA損傷
★ 同源重組
關鍵字(英) ★ 4-Aminobiphenyl
★ miR-630
★ MCM8
★ DNA Damage
★ Homologous Recombination
論文目次 中文摘要 I
英文摘要 II
誌 謝 III
目 錄 IV
圖目錄 VII
表目錄 VIII
第一章、 緒論 1
1-1 研究背景 1
1-1-1 4-胺基聯苯(4-aminobiphenyl;4-ABP) 1
1-1-2 4-胺基聯苯造成DNA損傷 1
1-1-3 4-胺基聯苯的致癌性 2
1-1-4 氧化壓力(Oxidative stress) 3
1-1-5 DNA損傷反應 (DNA damage response; DDR) 3
1-1-6 miRNA (microRNA; 微小RNA) 4
1-1-7 miRNA 的生成 4
1-1-8 miRNA對標靶基因mRNA的辨識與預測 6
1-1-9 環境毒素對於DNA修復蛋白之表觀遺傳調控 7
1-1-10 4-胺基聯苯所誘導的表觀遺傳學調控機制 7
1-2 研究目的 9
1-3 研究架構 9
第二章、 材料與方法 10
2-1 實驗材料 10
2-1-1 藥品 10
2-1-2 抗體 11
2-1-3 套組試劑 11
2-1-4 溶液製備 11
2-2 實驗方法 13
2-2-1 細胞株來源及細胞培養 13
2-2-2 細胞計數與存活測試 14
2-2-3 化學物質處理 15
2-2-4 細胞存活率分析 15
2-2-5 DNA損傷測定-彗星試驗 15
2-2-6 西方免疫墨點法 16
2-2-7 HepG2 RNA萃取 20
2-2-8 mRNA 反轉錄成cDNA 21
2-2-9 DNA及RNA 電泳 22
2-2-10 即時定量聚合酶連鎖反應 22
2-2-11 質體抽取 24
2-2-12 質體DNA及小片段RNA轉染 25
2-2-13 免疫螢光染色 26
2-2-14 同源重組活性測試 26
第三章、 結果 28
3-1 4-胺基聯苯誘導HepG2細胞造成細胞毒性 28
3-2 4-胺基聯苯誘導HepG2細胞DNA損傷 28
3-3 4-胺基聯苯對於miRNA-513a-5p 與miRNA-630的表現量的影響 28
3-4 4-胺基聯苯對於MCM8及MCM9之mRNA及蛋白質表現量的影響 29
3-5 miRNA-630會抑制MCM8表現量 29
3-6 ROS會誘導miRNA-630生成 30
3-7 MCM8表現量對於同源重組活性的影響 30
第四章、 討論 32
4-1 4-胺基聯苯對於HepG2細胞造成的毒性 32
4-2 DNA損傷誘導miRNAs生成 33
4-3 miRNA-630對DNA修復蛋白MCM8的調控 34
4-4 MCM8對於同源重組活性的影響 35
第五章、 結論 37
圖表 38
參考文獻 57
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指導教授 陳師慶(Ssu-Ching Chen) 審核日期 2018-6-28
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