柯逢春臺灣大學：分子與細胞生物學研究所鄧鈞鴻Teng, Chun-HungChun-HungTeng2007-11-252018-07-062007-11-252018-07-062004http://ntur.lib.ntu.edu.tw//handle/246246/49931粒線體為細胞之能量工廠，負責調控細胞內之能量生成與代謝途徑，改變粒線體之弁鄑Y可能影響整個細胞之命運，如細胞老化或Apoptosis。AOA(Aminooxyacetic acid)抑制Malate-Aspartate Shuttle使得Glycolysis產生之NADH還原力無法轉入粒線體，繼而影響TCA cycle之運行效率，造成細胞能量之耗損改變細胞內部代謝生理狀態。本研究顯示AOA的效應會誘發細胞老化，人類纖維母細胞WI-38持續處理2.5 mM AOA 2天後細胞老化比例開始明顯增加，隨時間增加細胞老化比例持續明顯增加，第六天高達68.5 %。為探討粒線體經由其他途徑誘發老化與AOA誘發老化之關係，因此利用其他如AICAR (5–Aminoimidazole- 4-carboxamide-1-b-D-ribofuranoside ；AMPK Activator)、NAC (N-Acetyl-L-cysteine ；ROS Inhibitor)與H2O2 (Hydrogen Peroxide)處理，研究其間可能之交互關係。同時處理1 mM AICAR與2.5 mM AOA 六天後，發現WI-38之老化比例並沒有加成作用，以100 μM NAC無法阻止 2.5 mM AOA所引發之細胞停止生長與細胞老化現象，顯示可能AOA所引發老化機制不是經由H2O2而是與AMPK的老化途徑重疊。由於AOA抑制Malate-Aspartate shuttle 可能影響粒線體內外的NAD+/NADH 平衡，所以測量細胞內之總NAD+/NADH ratio 之變化，實驗結果顯示處理後並無顯著之改變。此外，根據文獻記載，細胞Apoptosis時粒線體釋放AIF(Apoptosis-Inducing Factor)與細胞內的NAD+/NADH ratio變化可能相關，因此觀察AOA處理下AIF被釋放之狀況，在持續處理2.5 mM AOA引發老化條件下，由粒線體釋放之 AIF 量隨細胞老化之比例有增加之趨勢。Mitochondria, as cellular energy factory mediate producing energy and regulating metabolic pathway. However, mitochondrial dysfunction also affects the fate of cell, such as cellular senescence or apoptosis. Aminooxyacetic acid (AOA), a potent inhibitor of the mitochondrial malate-aspartate shuttle, causes cellular energy depletion by inhibiting the transfer of the NADH reducing potential produced by glycolysis to mitochondrion. In this study, we provide the evidence that AOA can also induce cellular senescence. By treating with 2.5 mM AOA, cellular senescence ratio began to increase at second day and up to 68.5% at sixth day. It is known that mitochondria dysfunction may result in AMPK activation or H2O2 generation. Both situations induced cellular senescence. In order to investigate AOA effect on cellular senescence has any relationship with the known pathways, cells were treated with 1 mM AICAR and 2.5 mM AOA simultaneously for six days. There is no additive effect in AOA-induced senescence ratio, and 100μM NAC(N-Acetyl-L-cysteine) which block H2O2 effect also can not prevent AOA effect on cellular senescence. AOA may influence the balance of NAD+/NADH between mitochondria and cytosol. By measuring total NAD+/NADH ratio, it shows no significant change. According to literature, during apoptosis process AIF released from mitochondria may be related to NAD+/NADH ratio. We did observe AIF released from mitochondria under 2.5 mM AOA treatment and it correlates to cellular senescence ratio.目錄 Ⅰ 圖表目錄 Ⅲ 簡寫名詞對照表 Ⅴ 中文摘要 Ⅵ 英文摘要 Ⅶ 引言 1 壹、細胞老化簡介 1 貳、粒線體與細胞老化之關係 3 一、ROS與老化 3 二、Ca2+與老化 4 三、AMP/ATP與老化 5 四、NAD+/NADH 與老化 6 参、細胞老化與細胞週期控制系統 9 肆、實驗目的 11 材料與方法 13 壹、試劑結構 13 貳、細胞培養 14 參、生長曲線 14 肆、SA-βGal 染 Senescence Cell 15 伍、NAD+/NADH 比例測定 16 陸、蛋白質萃取 17 柒、蛋白質電泳與免疫轉漬 17 捌、統計 18 結果 20 壹、持續處理2.5 mM AOA抑制WI-38生長 20 貳、持續處理2.5 mM AOA引發WI-38明顯老化 20 參、持續處理1 mM AICAR抑制WI-38生長並催發細胞老化 21 肆、持續同時處理2.5 mM AOA與1 mM AICAR六天並無法加成WI-38之老化 21 伍、處理100 mM NAC可抑制 250 μM H2O2所引起之WI-38老化但無法阻擋2.5 mM AOA所引起之老化現象 22 陸、持續處理2.5 mM AOA 無法顯著改變WI-38 之total NAD+/NADH Ratio 22 柒、持續處理 2.5 mM AOA增加 Mature AIF 表現 23 討論 24 參考文獻 28en-US纖維母細胞WI-38AIFAOAother