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作者(中文):林珮蓉
作者(外文):Lin, Pei-Jung
論文名稱(中文):砷阻礙細胞自噬作用進而誘發細胞凋亡之機制探討
論文名稱(外文):Arsenic treatment inhibits cell autophagy and leads to the occurrence of apoptosis.
指導教授(中文):林立元
指導教授(外文):Lin, Lih-Yuan
口試委員(中文):李易展
楊培銘
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:102080539
出版年(民國):104
畢業學年度:104
語文別:中文
論文頁數:71
中文關鍵詞:細胞自噬細胞凋亡
外文關鍵詞:autophagyapoptosisArsenic
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砷為常見的環境汙染物之一,許多研究指出砷造成的細胞毒性多是透過產生氧化壓力、粒線體膜電位衰減亦或是影響細胞週期誘發細胞凋亡。本篇研究主要探討砷的刺激造成人類胚胎腎細胞(HEK 293 cell)死亡的機制。在砷的刺激下,PI3K/Akt 訊號傳遞路徑會被抑制因而誘發cytochrome c由粒線體釋出,活化caspase 3導致細胞凋亡。然而PTEN不參與調控PI3K/Akt 訊號傳遞路徑,並且它的表現量隨著砷濃度增加而下降。進一步我們發現砷的處理,PTEN存在蛋白質緊密聚集體而不是被降解,同時幫助蛋白質正確折疊與修飾的熱休克蛋白70也會形成蛋白質緊密聚集體。另一方面,砷的刺激會誘發細胞自噬,同時溶酶體活性會被抑制。因此我們處理溶酶體活化劑(rapamycin),再給予砷的刺激,發現PTEN蛋白質緊密聚集體減少並且在NP-40 soluble fraction表現量下降,代表砷的刺激會抑制溶酶體活性阻礙細胞自噬的進行,促使PTEN蛋白質累積於autophagosome而不被降解。最後,砷與rapamycin共同處理,改善砷抑制PI3K/Akt 訊號傳遞路徑的情形,以致於細胞得以存活。綜合實驗結果,我們發現砷藉由抑制溶酶體活性阻礙細胞自噬的過程進而抑制PI3K/Akt 訊號傳遞路徑導致粒線體調控的細胞凋亡。
Arsenic is one of the environmental pollutants and a well-known carcinogen. Previous studies indicated that cytotoxicity caused by arsenic is recognized through generating oxidative stress, attenuating mitochondrial membrane potential, or influencing cell cycle to lead apoptosis. This study investigates the mechanism involved in the arsenic-induced cell death in Human Embryonic Kidney 293 (HEK 293) cells. Upon arsenic stimulation, PI3K/Akt signaling pathway is inhibited and leads to cytochrome c release from mitochondria which activates caspase 3 and apoptosis. However, PTEN is not participated in the PI3K/Akt signaling pathway. PTEN expression is reduced by arsenic treatment. Noticeably, the loss of PTEN is not due to the degradation of the protein, but presents in the insoluble fraction of the cells. The factor that assists protein refolding, heat shock protein 70 (Hsp 70), was also found in insoluble fraction. Concurrently, arsenic treatment inhibited the occurrence of autophagy in HEK 293 cells and caused an accumulation of LC3 in the cells. Administration of lysosomal activator (rapamycin) reduces the PTEN level in either soluble or insoluble fraction of the cells. The blockade of autophagy via reducing lysosomal activity caused the reduction of PI3K/Akt activity and led cells to apoptosis. Addition of rapamycin activates lysosomal activity and allows the cells to go through autophagy pathway. Our results reveal that arsenic treatment inhibits the lysosomal activity, and thus blocks the autophagy pathway. This blockade further reduces the activity of PI3K/Akt signaling pathway and leads to mitochondria-mediated apoptosis.
目錄……………………………………………………………………..3
中文摘要……………………………………………………………… 4
英文摘要……………………………………………………………….5
緒論……………………………………………………………………..6
材料與方法…………………………………………………………17
結果…………………………………………………………………….25
討論…………………………………………………………………….38
參考資料…………………………………………………………… 46
附圖…………………………………………………………………….52
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