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DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 康照洲 | |
dc.contributor.author | Ya-Ting Li | en |
dc.contributor.author | 李雅婷 | zh_TW |
dc.date.accessioned | 2021-06-08T05:02:27Z | - |
dc.date.copyright | 2010-09-09 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-08-19 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23494 | - |
dc.description.abstract | 多環芳香烴(PAHs)為廣泛存在的環境污染物,3-甲基膽蒽(3-methylcholanthrene,3-MC)屬於多環芳香烴中的一員,當3-甲基膽蒽活化多環芳香烴受體(aryl hydrocarbon receptor, AhR)後會與芳香烴受體核轉位蛋白(AhR nuclear translocator, ARNT, HIF-1β)形成複合體然後啟動下游代謝解毒酵素CYP1A1 等基因的轉錄活化。缺氧反應元件(hypoxia-responsive element, HRE)主要是藉由缺氧誘導因子-1α(hypoxia-inducible factor-1 alpha, HIF-1α)所調控,然而過去研究發現利用小鼠Cl41 細胞處理多環芳香烴或其衍生物會使HRE下游基因血管內皮成長因子(vascular endothelial growth factor, VEGF)的mRNA 表現上升,此外文獻也觀察到3-MC 處理小鼠primary hepatocytes 後並不會對缺氧誘導的基因造成改變。因此本篇目的欲探討多環芳香烴影響細胞HRE 活性的相關機制,本研究採用三種不同的細胞株作為實驗材料293T、GBM 及HepG2,進行暫時性轉染HRE reporter gene 後,分別處理不同濃度的3-甲基膽蒽後藉由HRE promoter reporter assay 觀察其活性變化,結果顯示293T 和GBM 細胞的HRE 具有活化的現象而在HepG2細胞中則否。而實驗進一步發現HepG2 細胞的AhR:ARNT 比值遠大於293T 和GBM細胞,然而此差異為AhR 蛋白的含量不同而造成的,因此本研究假設3-MC 造成此三株細胞的HRE 活性變化的現象可能與AhR 相關。此外利用RT-PCR 觀察293T 與HepG2細胞處理3-MC後其HRE下游基因VEGF 的mRNA表現量,在293T 細胞中VEGF mRNA表現量會上升而HepG2 細胞中則否;並且在西方墨點法分析中,發現293T 及GBM 細胞處理3-MC後並不會造成其HIF-1α 蛋白產生穩定累積的現象。本研究進一步利用AhRshRNA 使HepG2 細胞的AhR 靜默(silence)探討其扮演的相關角色,實驗發現AhR 靜默的HepG2 細胞處理三甲基膽蒽後,能誘導其HRE 活性上升,並且在與缺氧狀態下處理三甲基膽蒽後其HRE活性反應更強烈;而利用免疫沉澱法觀察發現AhR靜默的HepG2 細胞其HIF-1α與ARNT之間的鍵結力,而造成細胞中HRE活性改變。 | zh_TW |
dc.description.abstract | Polycyclic aromatic hydrocarbons (PAHs) are common environmental contaminants. 3-Methylcholanthrene (3-MC) belongs to a group of PAHs which is an aryl hydrocarbon receptor (AhR) agonist. 3-MC binds to AhR and subsequent translocates to the nucleus to activate the downstream gene expression, such as CYP1A1. Hypoxia-responsive element (HRE) are activated mainly by hypoxia-inducible factor-1alpha (HIF-1α) under hypoxia condition, but the present study found that exposure of cells to PAHs and its derivatives led to marked induction of VEGF in Cl41 cells, and other study in mice primary hepatocytes have shown that exposure to PAHs, 3-MC, did not significantly alter HIF target gene expfected hypoxia reporter) and increased HRE downstream gene VEGF mRNA level in 293Tression. In this study, we found that 3-MC can induce HRE activity (a transiently trans and GBM cells, but 3-MC had no effect on a transiently transfected hypoxia reporter in HepG2 cells. We also found that 3-MC-induced HRE activity in GBM and 293T cells is independent of HIF-1α. We observe HepG2 cells has a high level of AhR protein, so we thought that the differences may be due to the differences of AhR : ARNT ratios in the cells. In addition, we used AhR shRNA to silence the AhR of HepG2 cells, and we subsequent found that 3-MC can induced HRE activity in HepG2 AhR knockdown cells, and 3-MC can enhance HRE activity in HepG2 AhR knockdown cells under hypoxia. Besides, we also used immunoprecipitation assay to observe AhR, HIF-1α and ARNT interaction. In this experiment we observed that the AhR can inhibited HIF-1α-ARNT interactions. Therefore, the results suggest that AhR may affect HRE activity due to the decreased availability of ARNT for HIF-1α dimerization. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T05:02:27Z (GMT). No. of bitstreams: 1 ntu-99-R97447006-1.pdf: 3089785 bytes, checksum: 30ba7cf5f7385ce73cb391b95edf6bb1 (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | 口試委員審定書
謝誌 縮寫表(Abbreviations)………………………………………………………………i 圖表目錄(Figures and Tables)………………………………………………………ii 中文摘要………………………………………………………………….....................iii 英文摘要………………………………………………………………………………..iv 第一章 緒論(Introduction) 1.1 文獻回顧 1.1.1 多環芳香烴(Polycyclic aromatic hydrocarbons, PAHs)與三甲基膽蒽 (3-Methylcholanthrene, 3-MC)………………………………………………....2 1.1.2 多環芳香烴受體(Aryl hydrocarbon receptor, AhR)…………………………..4 1.1.3 缺氧誘導因子(Hypoxia inducible factors, HIFs)與缺氧反應元件 (Hypoxia-responsive element, HRE)…………………………………………..8 1.1.4 AhR 和HIF-1α與芳香烴受體核轉位蛋白(aryl hydrocarbon nuclear translocator, ARNT, HIF-1β)三者之關係……………………………………..11 1.2 研究動機…………………………………………………………………………..13 第二章 實驗材料與方法(Materials and Methods) 2.1 實驗材料 2.1.1 細胞株(Cells)…………………………………………………………………….15 2.1.2 藥品與試劑(Chemicals and Reagents)………………………………………..15 2.1.3 抗體(Antibodies)……………………………………………………………….17 2.1.4 質體(Plasmids)…………………………………………………………………..17 2.2 實驗方法 2.2.1 細胞培養(Cell culture)…………………………………………………………17 2.2.2 缺氧系統培養箱(Hypoxia chamber)…………………………………………...18 2.2.3 細胞毒性測試(Cell viability test/MTT assay)……………………………….18 2.2.4 細胞總蛋白質液收集(Cell lysate collection)…………………………………19 2.2.5 西方墨點法(Western blot analysis)…………………………………………..19 2.2.6 免疫沉澱法(Immunoprecipitation)…………………………………………..20 2.2.7 細胞RNA 萃取(RNA extraction)……………………………………………..21 2.2.8 反轉錄鏈鎖聚合酶反應(RT-PCR)…………………………………………..21 2.2.9 質體轉染(Plasmid transfection)………………………………………………22 2.2.10 電穿孔法(Electroporation)………………………………………………….23 2.2.11 Luciferase 冷光測定(Luciferase assay)……………………………………..23 2.2.12 生產重組慢病毒載體(Lentiviral vector)……………………………………24 2.2.13 病毒液感染與細胞篩選……………………………………………………….24 2.2.14 細胞核質分離(Nuclear and cytoplasmic fraction)…………………………...24 2.2.15 統計分析(Statistic analysis)…………………………………………………25 第三章 實驗結果(Results) 3.1 三甲基膽蒽及苯并芘的處理不會造成GBM、293T 和HepG2 產生細胞毒性...27 3.2 三甲基膽蒽及苯并芘對GBM、293T 和HepG2 細胞的缺氧反應元件(HRE) 活性的作用……………………………………………………...........................27 3.3 HepG2、GBM 和293T 細胞三者的多環芳香烴受體(AhR)和芳香烴核轉位 蛋白(ARNT)之表現量………………………………………………………….29 3.4 三甲基膽蒽藉由AhR 路徑所誘導的下游代謝基因CYP1A1 蛋白表現量在 HepG2中遠高於GBM和293T細胞……………………………………………..29 3.5 三甲基膽蒽在正常氧分壓與缺氧下影響HepG2 和293T 細胞的CYP1A1 與 VEGF 之mRNA 表現…………………………………………………………….30 3.6 293T 和GBM 細胞處理三甲基膽蒽後不會影響缺氧誘導因子-1α(HIF-1α)的 蛋白質表現量…………………………………………………………………….31 3.7 慢病毒載體(lentiviral vector)對HepG2 細胞的多環芳香烴受體的靜默 (silence)…………………………………………………………………………31 3.8 三甲基膽蒽對293T、HepG2 和多環芳香烴受體靜默的HepG2 細胞在正常氧 分壓與缺氧下之缺氧反應元件(HRE)活性的影響……………………..……32 3.9 多環芳香烴受體靜默後影響缺氧誘導因子-1α與芳香烴受體核轉位蛋白 (ARNT)的鍵結能力……………………………………………………………..33 第四章 討論(Discussion) 4.1 三甲基膽蒽(3-MC)與苯并芘(B[a]P)影響不同細胞內缺氧反應元件(HRE) 活性的差異性…………………………………………………………………...35 4.2 三甲基膽蒽的處理對293T 和GBM 細胞的HRE 活化現象並非透過使缺氧誘 導因子-1α穩定累積…………………………………………………………….37 4.3 在三甲基膽蒽影響細胞內缺氧反應元件活性中多環芳香烴受體(AhR)所扮 演的角色………………………………………………………………………...38 4.4 多環芳香烴受體(AhR)和缺氧誘導因子-1α之關連性………………………..40 第五章 結論(Conclusion)………………………………………………………..44 參考文獻(References)………………………………………………………………45 圖表集(Figures and Tables)…………………………………………………………61 | |
dc.language.iso | zh-TW | |
dc.title | 三甲基膽蒽對缺氧反應元件之影響:
多環芳香烴受體之角色探討 | zh_TW |
dc.title | Effects of 3-Methylcholanthrene on
Hypoxia-Responsive Element: The Role of Aryl Hydrocarbon Receptor | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 彭福佐,鄭幼文 | |
dc.subject.keyword | 三甲基膽蒽,缺氧反應元件,多環芳香烴受體,缺氧誘導因子-1α, | zh_TW |
dc.subject.keyword | 3-Methylcholanthrene (3-MC),hypoxia-responsive element (HRE),aryl hydrocarbon receptor (AhR),hypoxia-inducible factor-1alpha (HIF-1α), | en |
dc.relation.page | 78 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2010-08-19 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 毒理學研究所 | zh_TW |
顯示於系所單位: | 毒理學研究所 |
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