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研究生:駱紀東
研究生(外文):Ji Dung Luo
論文名稱:探討黃嘌呤氧化還原酶及尿酸對細胞抗氧化壓力能力所扮演的角色
論文名稱(外文):Role of Xanthine Oxidoreductase and Uric Acid Production in the modulation of Cellular Anti-oxidative Capability
指導教授:邱全芊
指導教授(外文):C. C. Chiou
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:55
中文關鍵詞:尿酸氧化壓力黃嘌呤氧化還原酶抗氧化物
外文關鍵詞:uric acidxanthine oxidoreductaseoxidative stressantioxidnat
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尿酸是人類嘌呤代謝的最終產物,也是血液中最重要的抗氧化物。製造尿酸的關鍵酵素,黃嘌呤氧化還原酶 (xanthine oxidoreductase, XOR),已證實會受到機械力(mechanical stress)、或缺氧狀況(hypoxia)等環境壓力所誘發。在此研究中,我們假設XOR可受到氧化壓力影響而誘發,增加尿酸產量。且此增加的尿酸量可供給細胞對抗氧化壓力。為了證明此論點,我們將細胞處理過氧化氫(hydrogen peroxide, H2O2),再以分光光度計測量細胞內尿酸含量。發現K562細胞經30分鐘處理後自5.46±0.57 μmol/mg proteins 增加到8.58±0.62 μmol/mg protein (p<0.01);TSGH細胞則自40.9±1.73 μmol/mg protein上升到52.8±1.64 μmol/ mg protein (p<0.01)。以real time RT-PCR及Western blot測量H2O2處理之後,XOR的mRNA及protein表現量變化。結果發現XOR mRNA和protein表現量並未隨著H2O2處理而增加。然而,XOR活性在處理H2O2後,卻有增加的情況。H2O2處理30分鐘之後,不管是K562或TSGH細胞的XOR activity 皆呈現明顯上升的情形 (p<0.01)。此外先以allopurinol,一種XOR的抑制物,處理細胞兩小時,抑制細胞內XOR活性,再處理H2O2,最後以MTT assay測量細胞存活率。結果發現以allopurinol處理之後的AGS細胞存活度自77.3±1.26% 降至70.1±1.81% (p<0.05);TSGH細胞的存活度則自86.0±0.72%降至81.2±0.45% (p<0.05)。總結以上結果,我們發現了一個經由提升XOR活性與增加尿酸產量卻不影響XOR基因表現量的抗氧化機轉。
Uric acid is the end product of human purine metabolism and acts as a major antioxidant in body fluid. Xanthine oxidoreductase (XOR), the key enzyme of uric acid synthesis, has been shown to be induced by environmental stress, such as mechanical stress and hypoxia. We hypothesize that oxidative stress could also activate XOR activity and increase uric acid production, which would enhance antioxidative ability of the cells. To prove this hypothesis, we challenged cells with H2O2 and measured uric acid amount with uricase-based spectrometric assay. The result showed that uric acid level in K562 cells after 2-hour H2O2 treatment was elevated from 5.46±0.57 μmol/mg proteins to 8.58±0.62 μmol/mg protein (p<0.01); uric acid in TSGH after 20-minute H2O2 treatment was elevated from 40.9±1.73 μmol/mg protein to 52.8±1.64 μmol/ mg protein (p<0.01). Real time RT-PCR and Western blot showed that XOR mRNA level and protein level did not change in K562 and TSGH after H2O2 treatment. However, XOR activity significantly increased after 30-minute H2O2 treatment in both K562 and TSGH cells (p<0.01) In addition, using allopurinol to inhibit XOR activity, cell survival rate decreased from 77.3±1.26% to 70.1±1.81% of untreated control in AGS (p<0.05) and from 86.0±0.72% to 81.2±0.45% of the untreat control in TSGH (p<0.05). In summary, we have found a possible antioxidative stress mechanism through increasing XOR activity and uric acid production but without altering XOR gene expression.
Contents
指導教授推薦書
口試委員會審定書
授權書 iii
致謝 iv
中文摘要 v
Abstract vii
Abbreviation ix
Contents xi
Table of Contents xiv

Chapter I Introduction 1
1.1 Uric acid 1
1.2 Uric acid maintains blood pressure in low-salt diet. 2
1.3 Uric acid is an inducible signal molecule. 3
1.4 Uric acid is an antioxidant. 3
1.5 Xanthine oxidoreductase 5
1.6 Regulation of xanthine oxidoreductase 6
1.7 Hypothesis 8
1.8 Specific aims 8
Chapter 2 Materials and methods 10
2.1 Cell line and chemicals 10
2.2 H2O2 treatment 11
2.3 MTT assay 11
2.4 Measurement of uric acid concentration 12
2.5 RNA extraction 13
2.6 Real time RT-PCR 14
2.7 Western blot 15
2.8 Detection of xanthine oxidoreductase activity 18
2.9 Statistics 19
Chapter 3 Results 20
3.1 Uric acid protected cells from H2O2-induced cell death. 20
3.2 Uric acid production was induced in both K562 and TSGH cells after 2-hour treatment with H2O2. 20
3.3 XOR mRNA level did not change in K562 and TSGH cells after H2O2 treatment. 21
3.4 H2O2 treatment did not alter XOR protein expression. 21
3.5 H2O2 treatment altered XOR activity. 22
3.6 Inhibition of XOR decreased cell survival rate after H2O2 treatment. 23
Chapter 4 Discussion 24
References 29
Figures 34





Table of contents
Figure 1: The flow chart of experimental design 34
Figure 2: Uric acid increased cell survival rate after treatment with hydrogen peroxide. 35
Figure 3: H2O2 induced uric acid production. 36
Figure 4: XOR mRNA level did not change after 2-hour H2O2 treatment. 37
Figure 5: XOR protein level did not change after 2-hour H2O2 treatment. 38
Figure 6: XOR activity increased after H2O2 treatment. 39
Figure 7: Inhibition of XOR activity decreased cellular anti-oxidative stress ability. 40
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