柯逢春臺灣大學：分子與細胞生物學研究所李俊陞Lee, Chun-ShengChun-ShengLee2007-11-252018-07-062007-11-252018-07-062005http://ntur.lib.ntu.edu.tw//handle/246246/49920粒腺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，改變粒腺體之功能即可能影響整個細胞之命運，如細胞老化或細胞凋零。我們使用AOA（aminooxyacetic acid）抑制malate-aspartate shuttle使得glycolysis產生之NADH還原力無法進入粒腺體，繼而影響TCA cycle運轉的效率，造成細胞能量之耗損改變細胞內部代謝生理狀態。 本實驗顯示以AOA處理人類正常纖維母細胞WI38會誘發細胞老化。以2.5mM AOA持續處理在6天後比例可以高達73.9%；然而在相同條件的處理之下，缺乏p16以及ARF的硬骨癌細胞U2OS並無細胞老化現象。另外在調節細胞週期進展分子上面，WI38細胞內p53、p21、p16逐漸累積，而pRb的狀態則顯示細胞週期停止。在細胞能量狀態上，我們觀察到[AMP]/[ATP]的比例隨著處理天數而上升，顯示細胞在能量的狀態逐漸改變。而細胞能量的缺乏亦反應在TOR complex以及S6K上。 AOA影響老化的效應可能有二：第一是影響非必需氨基酸之間的平衡、第二是影響粒腺體內能量的狀態。關於前者我們以外加非必需氨基酸的方式，試圖反轉AOA對於WI38的老化誘發，但是並沒有阻擋老化的產生。而後者，我們以TCA cycle 的中間產物 2-oxoglutarate（2-OG）與AOA共同處理，則可以明顯反轉AOA的老化，而且此反轉現象再以鐵離子的chelator DFO處理則會消失。同時，以抑制HIF-1α proline hydroxylation的藥劑也會造成細胞老化。本研究顯示AOA影響老化可能是透過2-OG的缺乏，進而累積HIF-1α的路徑來執行。Mitochondria, as a cellular energy factory mediates energy production and metabolic pathway regulation. Mitochondria 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 NADH reducing potential produced by glycolysis to mitochondria. This study, we provide the evidence that AOA can induce senescence in normal human fibroblast WI38. By treating with 2.5mM AOA, cellular senescence reached to 73.8% at the sixth day; in contrast, the same treatment could not induce cellular senescence in osteoblastoma U2OS cells, which lacks p16 and ARF. In addition to morphological phenotype, there is accumulation of p53, p16, p21 and retinoblastoma protein (Rb) is at the state of cell cycle arrest. Also, we observed the ratio of [AMP]/ [ATP] increased indicating a change of energy. It also reflects on the TOR and S6K signaling pathway. The mechanism of AOA induction of senescence may involve two pathways：One may act through the imbalance of non-essential amino acid; the other may through the energy state of mitochondria. For the former hypothesis, we tried to reverse the senescence by supplementing non-essential amino acid; however, no obvious effect. Then, we succeeded in reversing the AOA-induced senescence by co-treatment with 2-oxoglutarate (2-OG), the intermediate of TCA cycle, and co-treatment with Fe2+ chelator DFO abolished such reversion. In addition, chemicals inhibiting HIF-1α proline hydroxylation also induce senescence. Take together, this study indicates that AOA-induced senescence may mediate through the 2-OG and the HIF-1α pathway.摘要………………………………………………………………………1 英文摘要…………………………………………………………………2 引言………………………………………………………………………3 壹、 細胞老化簡介 A、 細胞老化形態及分子定義 B、 引發細胞老化的途徑 貳、 細胞週期調控系統 參、 細胞能量與老化 實驗目的………………………………………………………………17 材料方法………………………………………………………………18 壹、 細胞培養處理 貳、 生長曲線 參、 Senescence Associated β-galactosidase stain 肆、 [AMP]/[ATP]比例測定 伍、 細胞內涵物萃取 陸、 蛋白質電泳與西方轉漬法 結果……………………………………………………………………24 壹、 持續以AOA處理WI38細胞會引發細胞老化 貳、 持續以AOA處理U2OS細胞無法引發細胞老化 參、 持續以AOA處理WI38細胞會改變細胞能量狀態 肆、 持續以AOA處理WI38細胞改變mTOR pathway 伍、 對細胞供給非必需氨基酸無法明顯反轉AOA對於WI38細胞的老化 陸、 以2-OG處理可以反轉AOA對於WI38細胞的老化 討論……………………………………………………………………29 壹、 以AOA誘發細胞老化模式 貳、 WI38細胞與U2OS在AOA誘發老化機制上的差異 參、 AOA處理WI38引發細胞老化的探討 引用文獻………………………………………………………………47773392 bytesapplication/pdfen-US細胞老化AOAsenescenceotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/49920/1/ntu-94-R92b43019-1.pdf