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研究生:陳姿潔
研究生(外文):Zi-Jie Chen
論文名稱:氧化壓力對beta2-醣蛋白I基因調控之探討
論文名稱(外文):Effects of Oxidative Stress on beta2-Glycoprotein I Gene Regulation
指導教授:姜安娜姜安娜引用關係
指導教授(外文):An-Na Chiang
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:59
中文關鍵詞:beta2-醣蛋白過氧化氫氧化壓力
外文關鍵詞:beta2-glycoprotein IHydrogen peroxideoxidative stressAP-1NF-kBHuh7
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β2-glycoprotein Ι (β2-GPI)是一個主要由肝臟合成的血漿醣蛋白,許多文獻指出β2-GPI參與在凝血機制、三酸甘油酯代謝、抗磷脂症候群及動脈粥狀硬化的進程中,但對於β2-GPI可受何種刺激調控其基因表現所知仍相當有限。本研究主要在探討以過氧化氫使細胞內氧化壓力形成是否能調控β2-GPI表現以及其機制。實驗結果顯示過氧化氫可刺激Huh7細胞內β2-GPI mRNA和蛋白質表現量增加,並可促使β2-GPI分泌。許多研究指出氧化壓力可活化轉錄因子AP-1和NF-κB,而β2-GPI基因上游-2207到-2063有三個預測的AP-1結合位置,-1432/-1419有一個預測的NF-κB結合位置,且以西方墨漬法也證實過氧化氫可促使AP-1和NF-κB轉移至細胞核內表現量增加。利用電泳位移實驗分析則顯示,Huh7受過氧化氫的刺激可明顯增加核蛋白與AP1-2 (-2150/-2124)、AP1-3 (-2087/-2062) 和NF-κB (-1438/-1413)探針結合的能力。另分析β2-GPI轉錄活性變化可發現在過氧化氫的刺激下,含AP-1和NF-κB的reporter construct皆有顯著提升其轉錄活性,根據這些實驗結果可推測過氧化氫可能是藉由活化AP1和NF-κB因而增加β2-GPI轉錄活性。因此本篇研究開啟了調控β2-GPI基因表現機制的一個重要線索。
β2-glycoprotein Ι (β2-GPI) is a plasma glycoprotein primarily synthesized in the liver.
β2-GPI has been implicated in blood coagulation, triglyceride metabolism, antiphospholipid syndrome and atherosclerosis. However, the factors that influence β2-GPI expression still remain unclear. In this study, the effects of hydrogen peroxide on β2-GPI expression in Huh7 and its underlying mechanisms were investigated. The levels of β2-GPI mRNA, protein expression and protein secretion were increased in a dose-dependent manner after hydrogen peroxide stimulation. Numerous studies indicate that activator protein-1 (AP-1) and nuclear factor-κB (NF-κB) can be activated by oxidative stress. We found three putative AP-1 cis-elements located between -2207 and -2063, and one putative NF-κB cis-element at the region from -1432 to -1419 of the human β2-GPI promoter. Western blot analysis showed that hydrogen peroxide induced nuclear translocation of AP-1 and NF-κB. In addition, electrophoretic mobility shift assay revealed that hydrogen peroxide enhanced the binding of nuclear extracts to AP1-2 (-2150/-2124)、AP1-3 (-2087/-2062) and NF-κB (-1438/-1413). The promoter activity of β2-GPI gene which contains AP-1 and NF-κB cis-elements were raised by hydrogen peroxide stimulation. Taken together, these results suggest that hydrogen peroxide induced β2-GPI transcription activity in Huh7 via activation of AP-1 and NF-κB. This study provide an important clue in extend insights into the regulatory mechanisms of the β2-GPI gene expression.
目錄
內容 頁數
英文摘要 I
中文摘要 II
緒論 1
實驗材料 8
研究方法
細胞培養 14
Hydrogen peroxide 15
全RNA萃取 15
反轉錄反應 16
聚合酶連鎖反應 16
全細胞蛋白之萃取 16
十二烷基硫酸鈉-聚丙醯胺凝膠電泳分析 17
西方墨漬法 19
電泳位移分析實驗 21
限制酵素切割及連接反應 23
轉形作用 24
質體萃取 25 細胞轉染 25
Luciferase活性分析 26
β-galactosidase活性分析 27
細胞內活性氧分子之測定 28
細胞存活率分析 29
統計分析 29
實驗結果
過氧化氫對細胞存活率之影響 30
過氧化氫刺激下對β-GPI mRNA表現量的影響 230
過氧化氫刺激下β-GPI蛋白表現量及分泌量的影響 230 過氧化氫對AP-1及NF-κB轉移至核內表現量的影響 31
過氧化氫對核內AP-1及NF-κB結合於β-GPI基因上游結合序列之影響 231
過氧化氫對β-GPI基因上游AP-1及NF-κB結合序列之調控影響 233
討論 34
圖表 39
參考文獻 50
參考文獻
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