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標題: | Slug對乳癌細胞株MCF-7的影響 The effect of Slug in the MCF-7 breast cancer cell line |
作者: | Ming-Hung Hou 侯明宏 |
指導教授: | 陳燕惠(Yen-Hui Chen) |
關鍵字: | Slug,上皮間葉轉化,E-cadherin,ERα,GSK 3β,Nrf2,HO-1,MCF-7/WT, Slug,epithelial mesenchymal transition,E-cadherin,ERα,GSK 3β,Nrf2,HO-1,MCF-7/WT, |
出版年 : | 2015 |
學位: | 碩士 |
摘要: | 上皮間葉轉化 (epithelial mesenchymal transition,EMT) 對於癌細胞產生抗藥性及造成較差的預後有相當程度影響力。文獻指出,EMT過程的許多轉錄因子扮演重要的角色,如CDH1/2, ZEB1/2, Twist, Snail 1/2等。先前我們實驗室的研究發現在具 doxorubicin 抗藥性的MCF-7/ADR細胞株中表現出大量的Slug (Snail2) 蛋白質,而野生型MCF-7 (MCF-7/WT)則未偵測到。所以本研究主要探討外加Slug 對MCF-7/WT 細胞的影響。 首先利用反轉錄聚合酶連鎖反應技術,從 MCF-7/ADR 細胞中純化出Slug基因,再利用重組質體建構技術建構出帶有Slug 基因的質體,接著將質體利用轉染技術送入MCF-7/WT 細胞中瞬時大量表現,並以反轉錄聚合酶連鎖反應及西方墨點法方法觀察E-cadherin、ERα及抗氧化基因如Nrf2與HO-1的表現。 實驗結果顯示,大量表現Slug蛋白質的MCF-7細胞比對照組細胞具有更高的遷移能力,而上皮細胞標記因子,如E-cadherin、ERα的表現降低,並且具有較明顯的抗氧化基因如Nrf2、HO-1的表現,且有關細胞凋亡分子Bax、Bad亦受到Slug抑制,此結果顯示Slug的表現可能使細胞表現更多間質細胞特性的表徵。另外,細胞中加入不同濃度的doxorubicin,則大量表現Slug的細胞,其cleaved PARP的表現降低。文獻中指出GSK3β可調控下游Slug蛋白質的降解,所以本實驗利用GSK3β抑制劑 (LiCl) 來抑制GSK3β蛋白質,結果顯示LiCl與瞬時表現的Slug共同的存在下,能更快速促進上皮細胞標記因子,如E-cadherin、ERα的減少及促進抗氧化基因的表現。綜合所有結果顯示,Slug在誘導MCF-7/WT走向EMT中佔了重要的角色,未來Slug蛋白質在癌症治療上可以做為一個有效的標的。 Epithelial mesenchymal transition (EMT) is important for cancer cells progression and poor prognosis. Studies show that many transcription factors play important roles in EMT formation such as CDH1/2, ZEB1/2, Twist, Snail 1/2. Previous study from our labatory found that the doxorubicin-resistant breast cancer cell line MCF-7/ADR expressed high level of Slug (Snail2) protein expression, compared to the doxorubicin-sensitive breast cancer cell line MCF-7/WT. The aim of this thesis was to study the effect of Slug in MCF-7/WT cells. The full-length coding sequence of Slug was amplified from MCF-7/ADR using RT-PCR. Then the cloning technique was applied to construct the recombinant plasmid pSlug. The pSlug was transiently transfected into MCF-7/WT cells and the expression of E-cadherin, ERα and antioxidant genes Nrf2 and HO-1 were detected. Data revealed that in the Slug-overexpressed MCF-7/WT cells, the migration ability increased. The epithelial marker E-cadherin and ERα were downregulated and the antioxidant genes Nrf2 and HO-1 were upregulated, compared to control MCF-7/WT. The expression of apoptotic factors Bax and Bad was also downregulated in the Slug-overexpressed MCF-7/WT. On the other hand, the level of cleaved PARP protein decreased in the MCF-7/WT which transiently overexpressed Slug, accompanied with doxorubicin treatment. Since the Slug stability was regulated by GSK3β, the GSK3β inhibitor (LiCl) slightly stabilized Slug and accelerated downregulation of the expression of E-cadherin and ERα and upregulated the antioxidant genes Nrf2 and HO-1 in MCF-7/WT cells. Taken together, Slug may play an important role in EMT development in MCF-7/WT, and could be a crucial factor in cancer treatment in the future. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/52249 |
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顯示於系所單位: | 藥學系 |
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