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研究生:楊喻琇
研究生(外文):Yu Hsiu Yang
論文名稱:葡萄糖六磷酸去氫酶對NADPH與NADPH製造及NADPH依賴性酵素的影響
論文名稱(外文):Effects of Cellular Glucose-6-phosphate Dehydrogenase on NADPH, NADPH-Generating Enzymes and NADPH-Dependent Enzymes in HepG2 Cells
指導教授:趙崇義趙崇義引用關係
指導教授(外文):D.T.Y. Chiu
學位類別:碩士
校院名稱:長庚大學
系所名稱:醫學生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
論文頁數:57
中文關鍵詞:葡萄糖六磷酸去氫酶
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葡萄糖六磷酸去氫酶(Glucose-6-phosphate dehydrogenase, G6PD)之主要生化功能是產生NADPH;NADPH乃細胞內極為重要的輔酶及還原劑,可保護細胞不受氧化傷害。除了G6PD之外,細胞仍有其他產生NADPH的酵素幫助維持細胞內NADPH之含量,如isocitrate dehydrogenase (ICDH) 和 malic enzyme (ME)。而Thioredoxin系統則是利用thioredoxin reductase(TrxR)氧化NADPH達到保護細胞的效果。為探討究竟G6PD缺乏如何影響NADPH乃至於ICDH、ME與TrxR,我們使用RNAi的技術成功建立了G6PD knockdown的HepG2細胞株,發現G6PD knockdown細胞(Gi)內NADPH與NADP+比值約只有對照細胞的60%,且Gi細胞有較高的ROS。以活性測試或西方墨點法進一步探討ICDH, ME以及TrxR的表達,發現正常培養的情況下,ICDH、ME與TrxR的含量並未隨著G6PD降低而改變。由於G6PD缺乏將導致ROS上升,故我們以氧化劑diamide來研究G6PD缺乏對氧化劑的反應,由細胞存活率可知Gi細胞較容易受到diamide影響而死亡,而diamide作用在G6PD正常細胞可引發G6PD、ICDH之活性上升,由此推論G6PD的確會影響NADPH產生,但目前之實驗結果顯示,在G6PD缺乏之情況下,細胞沒有明顯調整其他NADPH-generating酵素之表達,也並未影響TrxR之表達。
One of the major biological functions of glucose-6-phosphate dehydrogenase (G6PD) is to generate NADPH, which is an extremely important reductant, protecting cells from oxidative damage. There are still other NADPH generating enzymes to preserve cellular NADPH content, such as isocitrate dehydrogenase (ICDH) and malic enzyme (ME). On the other hand, thioredoxin (Trx) cascade delivers electrons from NADPH to the substrate protein via Trx and thioredoxin reductase (TrxR). How G6PD deficiency may affect NADPH generation and the expression of ICDH, ME as well as TrxR have not been delineated. To investigate these questions, G6PD expression was knocked down in HepG2 cells, using RNAi technique. NADPH content in G6PD knockdown cells was about 60% of that in control cells. Moreover, G6PD knockdown cells exhibited higher level of reactive oxygen species (ROS). In basal culture condition, there was no significant difference in the expression of ICDH activity as well as ME between G6PD knockdown and control cells and so does TrxR protein expression. Since G6PD-knockdown can increase cellular ROS level, diamide, an oxidant, was used to investigate how such treatment can affect G6PD knockdown cells. Using viability test, G6PD knockdown cells were found to be more sensitive to diamide induced cell death. Furthermore, G6PD and ICDH activity was upregulated in G6PD control cells. Taken together, these findings suggest that G6PD can affect NADPH generation. However, G6PD knockdown did not affect the expression of other NADPH generating enzymes nor TrxR under our current experimental conditions.
指導教授推薦書…………………………………………………………
口試委員會審定書………………………………………………………
授權書………………………………………………………………….iii
誌謝…………………………………………………………………….iv
中文摘要……………….………………………………………………v
英文摘要……………………………………………………………….vi
前言……………………………………………………………………1
材料與方法……………………………………………………………6
結果……………………………………………………………………15
討論……………………………………………………………………20
參考文獻………………………………………………………………25
圖表……………………………………………………………………36
圖一、確認建立出G6PD knockdown之細胞株………………………36
圖二、以UPLC偵測細胞中NADP+含量………………………………37
圖三、以UPLC偵測細胞中NADPH含量……………………………38
圖四、G6PD缺乏細胞中之ROS含量較高……………………………40
圖五、G6PD缺乏細胞之isocitrate dehydrogenase(ICDH)表現類似對照細胞……………………………………………………………………41
圖六、G6PD缺乏細胞之malic enzyme(ME)活性類似正常細胞……42圖七、G6PD缺乏細胞之thioredoxin reductase(TrxR)蛋白表現接近對照細胞…………………………………………………………………43
圖八、G6PD缺乏細胞對氧化劑diamide之敏感性高於正常細胞…44
圖九、Diamide處理提高正常細胞之G6PD活性表現………………45
圖十、Diamide處理亦提高正常細胞之ICDH活性表現……………46
圖十一、Diamide處理不影響ME活性………………………………47
圖十二、Diamide降低細胞GSH且正常細胞回復較快……………48
表一、G6PD缺乏細胞之NADPH及NADP+比值下降………………39
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