砷是環境中常見的汙染物質，在許多研究報告中指出，砷造成的細胞毒性會產生氧化壓力進而造成細胞凋亡。然而，最近有研究顯示細胞自噬參與在砷誘導的細胞反應。本篇論文主要在探討當人肺腺癌細胞 (A549 cell) 受到砷刺激後，p53 蛋白在細胞自噬 (autophagy) 與細胞凋亡 (apoptosis) 作用上所扮演的角色，並以不含有 p53 基因的非小細胞肺癌細胞 (H1299 cell) 來比較 A549 細胞中砷誘導的反應。首先我們發現0到100 µM的砷處理，不會對 A549 細胞的存活率造成顯著影響，但 H1299 細胞存活率會隨著砷濃度增加而明顯下降。我們也發現隨著處理砷的濃度增加，會誘發 A549 細胞中腫瘤抑制基因 p53 蛋白隨之增加。進一步我們發現當 A549 細胞中p53 蛋白被砷激活，進而提高 PTEN 表現，以抑制 PI3K/Akt/ mTOR 訊號傳遞路徑，促進細胞自噬作用的產生。接著我們在 A549 細胞中處理 NH4Cl 抑制細胞自噬和降低細胞活性，與砷共處理可以阻斷細胞自噬並活化細胞凋亡；另外使用自噬活化劑 rapamycin，可以緩解H1299 細胞中由砷造成的細胞凋亡。這些結果表示，細胞自噬能夠在砷造成的細胞損傷中發揮保護的作用，並抑制細胞凋亡的發生。另一方面，我們使用 PFT-α 來降低 p53 蛋白活性或以 siRNA 抑制 p53 基因表現，在砷的處理下則會阻斷細胞自噬路徑的活化，導致細胞凋亡增加。相反的，在 H1299 細胞中表現 p53 基因則會在砷處理下增加細胞存活率。我們的研究結果證明，p53 蛋白是調節砷誘導的細胞自噬的關鍵調節因子。p53 的存在可以激活細胞自噬並減少由砷所造成的細胞傷害。

Arsenic is a common environmental contaminant. Previous studies indicated that arsenic exposure increases oxidative stress, and leads cell to apoptotic death. However, autophagy was recently indicated to participate in the arsenic-induced cell responses. We investigated in this study the role of p53 in autophagy and apoptosis of A549 cells with arsenic treatment. A p53-null H1299 cell line was used to compare the arsenic-induced responses with A549 cells. Treating cells with 0 to 100 μM sodium arsenite did not cause significant effect on the viability of A549 cells. However, survival rate of H1299 cells decreased significantly with the increment of arsenite concentrations. Analysis of p53 revealed that the protein increased with the arsenite treatment in A549 cells. Activation of p53 increases PTEN expression which causes the inhibition of PI3K/Akt/mTOR signal transduction pathway to activate autophagy in A549 cells. Administration of NH4Cl in A549 cells inhibited autophagy and reduced cell viability. On the contrary, arsenic treatment blocked autophagy and led cell to apoptotic death in H1299 cells. Rapamycin, an autophagy activator, rescued H1299 cells from arsenite-induced apoptosis. These results indicate that autophagy protects cells form arsenic-induced cell damage and reduces the occurrence of apoptosis. We further used PFT-α to reduce p53 activity or siRNA to knockdown p53 gene expression with arsenite treatment. The fraction of apoptotic cell increased with the blockade of autophagy. Expression of p53 in H1299 cells otherwise increased the cell viability with arsenic treatment. Results from our study demonstrate that p53 is a key regulator to modulate the progression of arsenite-induced autophagy. Presence of p53 activates autophagy and reduces cell damages upon arsenite exposures.

中文摘要…………………………………………………………………2
英文摘要…………………………………………………………………4
緒論………………………………………………………………………6
材料與方法……………………………………………………………..17
結果……………………………………………………………………..26
討論……………………………………………………………………..36
參考文獻………………………………………………………………..42
附圖……………………………………………………………………..49

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