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作者(中文):張薰勻
作者(外文):Chang, Hsur-Yun
論文名稱(中文):AMP蛋白激酶的活化對金屬硫蛋白基因表現之影響
論文名稱(外文):Effect of AMP-activated Protein Kinase Activation on Metallothionein Gene Expression
指導教授(中文):林立元
指導教授(外文):Lin, Lih-Yuan
學位類別:碩士
校院名稱:國立清華大學
系所名稱:分子與細胞生物研究所
學號:9580579
出版年(民國):98
畢業學年度:97
語文別:中文
論文頁數:47
中文關鍵詞:金屬硫蛋白能量缺失
外文關鍵詞:AMPKMTenergy-deprivation
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金屬硫蛋白 (Metallothionein, MT)在生理上重要的功能為調節細胞內金屬的恆定性和對抗氧化壓力,而近幾年很多研究指出MT可能參與能量代謝的生理活動,但是仍沒有明確的機制。在研究中我們使用能量代謝抑制劑NaNB3B和2-deoxyglucose (2DG)處理HEK293細胞使之處於能量缺乏的情況,發現MT基因的表現受到抑制,且NaNB3B和2DG因阻斷細胞內ATP的生成而促進能量監督者AMPK的活性。由於AMPK調控細胞中能量的平衡,包括參與代謝的蛋白質或基因,於是我們測試NaNB3B是否透過活化AMPK而抑制MT基因的表現,我們利用Dominant-negative AMPK (DN-AMPK)與AMPK siRNA抑制AMPK的活性,發現當AMPK的活性被減低後,MT基因表現被NaNB3B抑制的情形減緩,故推測NaNB3B透過活化AMPK來調控MT基因的表現。當細胞遭受能量低迷的時候,活化的AMPK會藉由磷酸化受質來調控下游的代謝途徑與基因的表現,以維持細胞中的能量平衡,於是我們測試了與代謝有關的c-Myc和AMPK的受質Forkhead box O3 (FOXO3)是否參與在NaNB3B抑制MT基因表現的途徑中,結果顯示c-Myc和FOXO3均可能會影響MT基因表現,但不參與在NaNB3B活化AMPK而抑制MT基因表現的途徑。
中文摘要 I
Abstract II
目次 III
圖次 IV
緒論 1
材料與方法 8
1.化學藥品及製備 8
2.細胞株與培養條件 8
3.細胞質RNA萃取 (Extraction Cytoplasmic RNA) 9
4.甲醛變性瓊脂凝膠電泳 (Formaldehyde/denaturating agarose gel electrophoresis) 9
5.反轉錄 (reverse transcription) 10
6.即時定量聚合酶連鎖反應 (Quantitative Real-time PCR) 11
7.蛋白質定量 11
8.西方墨點法 (Western Blot) 12
9.轉殖作用 (Transfection) 14
10.報導基因檢定 (luciferase assay) 16
結果 17
1. NaN3抑制HEK293細胞的MT基因表現、且活化細胞中的AMPK 17
2. 2DG抑制HEK293細胞的MT基因表現、且活化細胞中的AMPK 17
3. NaN3透過活化AMPK來調控MT基因的表現 18
4.FOXO3不參與NaN3活化AMPK進而調控MT基因的途徑 21
5.c-Myc不參與NaN3活化AMPK進而調控MT基因的途徑 22
討論 25
參考文獻 33
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