臺灣博碩士論文加值系統

葡萄糖-6-磷酸脫氫酶 ( G6PD ) 是五碳糖磷酸化途徑 ( Pentose phosphate pathway ) 的第一個酵素，也是速率決定步驟。G6PD藉由調控還原態菸醯胺腺嘌呤二核酸磷酸 ( NADPH ) 的再生，維持細胞內還原態 ( GSH ) 與氧化態的穀胱甘肽 ( GSSG ) 之間的比值。我們利用RNAi的技術建立了G6PD knockdown的HepG2細胞 ( Gi ) 作為細胞模式去闡述在氧化壓力下GSH再生異常造成的補償機制，在正常生長情況下，Gi細胞NADPH / NADP+ and GSH / GSSG比值比對照組細胞 ( Sc ) 相對較低。當接觸到氧化劑diamide時，Gi細胞對於氧化劑的傷害較為敏感，且GSH的再生與GSSG的清除效率較差。另外這兩株細胞在diamide的處理下都呈現NAD+濃度降低與NADP+濃度增加的情況，但是Gi細胞的改變量比起Sc細胞更加顯著。我們還發現Gi細胞在diamide處理下，增強了NAD kinase的活性。雖然在G6PD knockdown細胞有代償性的NADPH濃度增加的情況，但是依然有GSH的再生與GSSG的清除效率較差的情況。因此我們推測G6PD藉由有效的GSH再生達到保護細胞對抗氧化壓力造成的傷害。

Glucose-6-phosphate dehydrogenase (G6PD), the first and rate-limiting enzyme of the pentose phosphate pathway, is important to maintain intracellular reduced and oxidized glutathione ( GSH / GSSG ) ratio via NADPH regeneration. G6PD knockdown HepG2 ( Gi ) cells by RNAi technique were used as a model to elucidate the compensatory mechanism for ineffective GSH regeneration in G6PD knockdown cells under oxidative stress. Under basal condition, intracellular levels of NADPH / NADP+ and GSH / GSSG ratio were lower in Gi cells than those in control ( Sc ) cells. When cells were exposed to diamide, Gi cells were more susceptible to diamide-induced cell death compared with Sc cells. GSH regeneration and GSSG clearance were ineffective in Gi cells. NADP+ level was increased and NAD+ level was decreased in both cells. However, the increase of NADP+ and the decrease of NAD+ were larger in Gi cells than those in Sc cells. We found that NAD kinase activity was increased in Gi cells more dramatically than Sc cells when cells were exposed to diamide. Although NADPH level was significantly increased in G6PD knockdown cells, which were with an impaired ability to regenerate glutathione after diamide treatment. Our findings suggest that G6PD confers protection against oxidant-induced cytotoxicity through effective glutathione regeneration.

目錄
指導教授推薦書………………………………………………………
論文口試委員會審定書………………………………………………
長庚大學授權書………………………………………………………iii
誌謝……………………………………………………………………iv
中文摘要………………………………………………………………v
英文摘要………………………………………………………………vi
目錄……………………………………………………………………vii
圖表目錄………………………………………………………………viii
前言……………………………………………………………………01
實驗目的………………………………………………………………07
實驗設計………………………………………………………………08
實驗材料與方法………………………………………………………09
實驗結果………………………………………………………………29
討論……………………………………………………………………35
參考文獻………………………………………………………………40
圖表……………………………………………………………………50
附錄……………………………………………………………………62


圖表目錄
圖一、實驗設計………………………………………………………50
圖二、確認G6PD knockdown細胞株之建立………………………51
圖三、G6PD knockdown HepG2細胞對於氧化劑diamide之敏感性大於對照細胞…………………………………………………52
圖四、G6PD knockdown Hep G2細胞在diamide處理下，GSH的回復速率與GSSG的清除速率都顯著較慢……………………53
圖五、G6PD knockdown細胞在diamide處理下其細胞內NADP+與NADPH含量顯著增加………………………………………54
圖六、G6PD knockdown細胞在diamide處理下其NAD kinase活性顯著增加………………..………………………………………56
圖七、G6PD knockdown細胞在diamide處理下，其isocitrate dehydrogenase和malic enzyme活性並無顯著改變……57
圖八、G6PD knockdown細胞在diamide處理下glutathione reductase活性並無顯著改變......................................................58
圖九、 G6PD knockdown細胞在diamide處理下其蛋白glutathionylation程度顯著增加...............................................59
圖十、根據實驗結果推論之可能機制圖…………………………61

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