亞砷酸鈉誘發人類臍靜脈內皮細胞Enolase及Fascin羰基化作用之探討

Effect of sodium arsenite induced carbonylation on Enolase and Fascin in human umbilical vein endothelial cells

林艾璇(Ai-Hsuan Lin)

指導教授 ： 李宗貴 ； 王祖興

中山醫學大學/健康管理學院/營養學研究所/碩士(2006年)

https://doi.org/10.6834/CSMU.2006.00053

摘要

工業污染及水源污染使人暴露於無機砷的環境，導致慢性砷中毒。流行病學研究證實慢性砷中毒與許多慢性疾病發生有關，其機轉與砷增加體內氧化壓力有關。活性氧造成胞內蛋白質氧化，改變蛋白質功能或影響訊號傳遞，使得細胞生理特性發生變化，最後產生病變。本實驗探討在亞砷酸鈉處理下，胞內蛋白質是否發生羰基化。人類臍靜脈內皮細胞（human umbilical vein endothelial cell）分別以0.5~50 μM 亞砷酸鈉、4 mM 過氧化氫處理30分鐘，再以二維電泳結合西方墨點法偵測羰基化蛋白質，經軟體比對及質譜儀序列分析，鑑定出參與醣解作用之酵素α-Enolase、肌動蛋白結合蛋白質Fascin及中間絲（intermediate filaments）蛋白質Vimentin是亞砷酸鈉誘發羰基化之標的蛋白。進一步分析Enolase酵素活性變化並進行胞內Fascin與Vimentin染色觀察。結果顯示：亞砷酸鈉處理以劑量依賴關係造成 Enolase 酵素活性降低，在50 μM砷處理下，胞內Fascin分佈由規則絲狀轉變成不規則分散狀，細胞形態也從紡錘狀變成圓形，且延伸出許多突刺。因此，推測砷經由增加胞內氧化壓力，使得胞內Enolse、Fascin與Vimentin發生羰基化修飾作用，進而降低細胞內醣解反應進行，並影響細胞架構蛋白穩定性。

關鍵字

砷 ； 蛋白質羰基化 ； 二維電泳 ； Enolase ； Fascin

並列摘要

The pollution of drinking water make people expose to the inorganic arsenic and cause the chronic arsenic poisoning. Many epidemiologic studies have demonstrated that the chronic arsenic exposure associated with the increasing of chronic diseases. Exposure of arsenic would induce ROS overload and result in protein oxidation. In this study, we examined the arsenite induced protein carbonylation and the functional effects of carbonylated proteins in human umbilical vein endothelial cells (HUVECs). By two-dimensional gel electrophoresis and matrix assist laser desorption ionization time of flight mass spectrometery(MALDI-TOF/MS) analyzed methods, three of arsenic-sensitive carbonylated proteins were identified to be the α-Enolase、Fascin and Vimentin. We further examined α-Enolase by enzyme activity assay. The results showed that arsenite decreased the Enolase activity in dose-dependent. In addition, we also observed that the arsenite would disturb the F-actin filament by carbonylation of Fascin. As these results, we suggested that the protein which was modified by arsenite would lead to protein carbonylation and further to effect physiological state of cells.

並列關鍵字

arsenite ； carbonylation ； 2D electrophoresis ； Enolase ； Fascin

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