博碩士論文 103821002 詳細資訊




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姓名 徐同緯(Tung-Wei Shu)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 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
(Molecular Mechanisms of Dicrotophos-Induced Toxicity in HepG2 cells:The Role of CSA in Toxicity)
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摘要(中) 底特松(Dicrotophos;Dic)是一種含有有機磷(Organophosphorous)化合物的農藥,是一種能夠快速且有效的消除蟲害且對環境有較低毒性被廣泛用於世界各地。本篇研究主要深入探討,底特松(Dicrotophos)所造成毒性機制包含氧化壓力及DNA損傷。彗星試驗(Comet assay)主要測定是否產生基因毒性可能性,結果發現處理底特松於(50-200µM) 24小時後導致HepG2 Cells 發生DNA damage,同時利用JC-1螢光染劑檢測粒線體膜電位變化,發現處理底特松於(50-200µM) 24小時後粒線體膜電位受到顯著性的抑制,然而在實驗中以抗氧化劑 5 mM N-acetyl-I-cysteine (NAC)預先處理細胞後,再以彗星試驗觀察發現DNA 斷裂程度,結果發現顯著性降低,粒線體損傷程度下降(p-value <0.05)。推測氧化物為造成DNA 斷裂程度原因之一。並以次世代定序(NGS) 的技術探討HepG2處理50µM底特松(Dicrotophos;Dic) 24小時後檢測所有gene表現量有差異的變化 (> or < 2-fold, p-value <0.05);由檢測結果得知底特松會影響Oxidative phosphorylation、Nucleotide excision repair及Cell cycle等相關傳遞路徑及其基因皆接受到顯著性的影響,而Oxidative phosphorylation及Nucleotide excision repair有18個及11個Gene受到顯著性的抑制,我們利用qRT-PCR再次驗證NGS的結果皆有符合趨勢,其中Nucleotide excision repair中上游關鍵之基因CSA最為顯著。因此想深入探討, DNA repair gene CSA的功能失調,是否造成DNA及粒線體損傷的主要原因。利用質體轉染技術,轉染24小時候讓細胞內的CSA大量表現,亦可發現DNA damage及Mitochondrial dysfunction 的程度皆獲得改善,並利用流式細胞儀驗證 Cell cycle arrest 與結果相互呼應,處理底特松(50-200µM) 24小時確實能夠誘導細胞週期停滯於G1期。
實驗結果顯示,我們發現底特松(Dicrotophos)的毒性機制能讓細胞誘導 ROS 生成並且抑制核苷酸切除修復Pathway中重要基因CSA、造成粒線體膜電位改變及 DNA damage 發生,而造成 DNA damage 的大量累積是因為DNA repair中CSA基因被抑制而無法適時的修復DNA所造成的,然而,上述基因的變化是否與底特松(Dicrotophos)誘導 HepG2 細胞生長的基因毒性需要更加深入探討。
摘要(英) Dicrothphos (Dic), an insecticide and acaricide, is used against a variety of sucking, boring and chewing pests. Dicrothphos has been proved to induce oxidative DNA damage in HepG2 cells in our previously published paper. However, the molecular mechanisms of this compound were still unclear. First of all, the cytotoxicity and oxidative DNA damage was confirmed. Next, we first used RNA-seq, a high- throughput method, to analyze the global expression of differential expressed genes (DEGs) in cells treated with 50 μM dicrothphos for 24 h, as compared to that in the control group (only 0.5 % DMSO). Results showed that these dysregulation of these genes irrespective of up or down regulation could be assigned to some diverse pathways/metabolisms using KEGG analysis, particularly in the DNA damage responses (DDR) such as oxidative phosphorylation, nucleotide excision repair, and cell cycle arrest. Validation of some randomly selected these DDR genes confirmed RNA-seq results. We further demonstrated that dicrothphos induced ROS overproduction, the loss of mitochondrial depolarization, and cell cycle arrest at G0/G1 phase. Interestingly, additional evidence revealed that DDR and apoptosis/autophagy could be alleviated in the presence of N-acetyl-cysteine (NAC), indicating that ROS-dependent DDR involvement in dicrothphos-induced cell death. In addition, we also definitely clarified the role of CSA, one enzyme of nucleotide excision repair enzymes, in oxidative DNA damage triggered by dicrothphos despite of its role in oxidative stress invoked by other oxidants was controversial. Our finding support the hypothesis that defective repair of oxidative DNA damage was at least partly involved in one of toxic mechanisms of dicrothphos against the growth of cells.
關鍵字(中) ★ Dicrotophos
★ Reactive oxygen species(ROS)
★ DNA damage
★ Next Generation Sequencing (NGS)
★ CSA
關鍵字(英)
論文目次 中文摘要 i
英文摘要 iii
第壹章、緒論 1
第貳章、實驗目的和實驗架構 12
第參章、材料與方法 13
第一節、實驗方法 13
一、細胞株來源及細胞培養 13
二、細胞計數 14
三、化學物質之處理 14
四、細胞存活率分析 15
五、細胞內活性氧化物質之測定 16
六、蛋白質萃取 19
七、蛋白質濃度測定 20
八、西方墨點法 21
九、RNA萃取 23
十、Messanger RNA反轉錄成cDNA 24
十一、DNA/RNA電泳 25
十二、即時定量聚合酶連鎖反應 26
十三、彗星試驗 27
十四、次世代定序技術(Next Generation Sequencing;NGS) 29
十五、大腸桿菌勝任細胞製備 30
十六、切開雙股DNA之限制酵素 31
十七、插入子與載體接合 32
十八、質體的抽取 34
十九、細胞週期分布之分析 35
二十、粒線體膜電位之檢測 36
第肆章、結果 38
第伍章、討論 44
參考文獻 53
圖表 63
附錄 88
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指導教授 陳師慶(Ssu-Ching Chen) 審核日期 2016-7-5
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