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研究生(外文):I-Wen Song
論文名稱(外文):The Functional Role of ARNT/HIF-1β in Cell cycle Regulation
指導教授(外文):Wen-Chang ChangBen-Kuen Chen
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在缺氧(hypoxia)的狀態下, Hypoxia inducible factor 1(HIF-1)是調控基因表現,幫助細胞度過難關最主要的轉錄因子。HIF-1因子有HIF-1α及HIF-1β/ARNT兩個成員,HIF-1α主要是在缺氧的時候表現,執行其功能;而ATNT在細胞中參與了胚胎發育、胰島β cell功能以及細胞生長的調控中,具有非常多元的角色。在我們實驗室先前的研究中發現,在normoxia的狀態下,ARNT參與在EGF處理下所誘導的cycloxygenase-2及12(S)-lpoxygenase基因表現中,這暗示著在normoxia的狀態下,ARNT可能參與在腫瘤細胞生長當中。於是,我們提出了一個假設,在固體腫瘤細胞生長的過程中,當腫瘤達到一定的大小時,便會呈現缺氧的情形,此時HIF-1α會表現,促使血管新生(angiogenesis)、醣類分解(glycolysis)以及細胞週期停滯(cell cycle arrest),幫助腫瘤細胞生存;然而在腫瘤剛形成的初始階段,是處在一個normoxia的狀態,此時ARNT是否透過調控下游的某些機制進一步調控了腫瘤細胞的生長?我們進一步利用proliferation assay分析這個問題。利用細胞計數及BrdU incorporation assay,在ARNT knockdown的HeLa細胞中,細胞的生長受到抑制。進一步我們分析了細胞週期中ARNT的表現量,發現在interphase時ARNT的表現量比在mitosis phase時來的低,更有趣的,在S/G2 phase的地方有一個劇烈減少的現象,而且,幫我們將ARNT knockdown後,S phase會有延長的現象。除此之外,在ARNT knockdown的HeLa細胞中,cyclin E的表現量受到抑制,這表示在normoxia狀態之下,ARNT可能是透過調控細胞週期中的因子如cyclin E,進而調控了細胞的生長。
Hypoxia-inducible factor 1 is an important transactivation factor which controls the expression of most genes under hypoxia condition. It is composed by two subunits: HIF-1α and HIF-1β/ARNT. As HIF-1αmainly plays its role under hypoxic condition, ARNT has been reported to involve widely in many cellular events, such as embryonic development,β-cell function regulation in type Ⅱ diabetes and cell proliferation. In our previous study, we found that ARNT was involved in EGF-induced cyclooxygenase-2 (COX-2) expression in normoxia condition. This finding indicates that ARNT may be involved in tumor growth under normoxia condition. We raise a hypothesis that when solid tumor size reach a certain degreed, it would be under a hypoxia condition which HIF-1α would likely to lead cells to survival by triggering angiogenesis, glycolysis and cell cycle arrest. Before that, in the initiate stage of solid tumor growth, will ARNT play a main role in mediate tumor growth under such normoxia condition? To further clarify whether ARNT was involved in the regulation of cell growth, we analyzed cell growth by proliferation assy. By cell number counting and BrdU incorporation assay, we found that cell growth rate was decreased in ARNT knockdown HeLa cell. We also analyzed ARNT level during cell cycle by synchronizing HeLa cells with nocodazole or thymidine. We found that ARNT level was lower in interphase as compare to mitosis phase. More interestingly, we found that ARNT level sharply decrease in S/G2 phase. More interestingly, ARNT knockdown extended S phase duration in HeLa cells. Furthermore, we also found that cyclin E decreased under ARNT knockdown HeLa cells. This indicates that ARNT may regulate cell cycle through cyclin E and play its role in cell growth control under normoxia.
中文摘要 ------------------------------------------------- Ⅱ
英文摘要 ------------------------------------------------- Ⅲ
附圖目錄 ------------------------------------------------- Ⅴ
縮寫指引 ------------------------------------------------- Ⅵ

第一章 緒論 ---------------------------------------------- 1

第二章 實驗材料及方法
來源 ------------------------------------------------------ 8
實驗材料及方法 -------------------------------------------- 14

第三章 實驗結果
第一節 ARNT與cell growth ---------------------------------- 23
(一) ARNT在tumor及normal cells中表現量的差異
(二) ARNT對cell growth的影響
(三) 排除HIF-1α的參與
第二節 ARNT 與cell cycle ----------------------------------- 24
(一) ARNT表現量在cell cycle中的變化
(二) ARNT在cellcycle中劇烈變化的stage
(三) ARNT knockdown對cell cycle progression的影響
(四) ARNT所影響的cell cycle factor

第四章 討論 ---------------------------------------------- 29
第五章 參考文獻 ------------------------------------------ 35
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