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系統識別號 U0026-2408201016131400
論文名稱(中文) 研究N-乙醯基半胱胺酸在檸檬素缺乏細胞之保護作用
論文名稱(英文) Study of the protective effects of N-acetylcysteine in citrin-deficiency cells
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系碩博士班
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 98
學期 2
出版年 99
研究生(中文) 劉秉芬
研究生(英文) Ping-Fen Liu
學號 t3697106
學位類別 碩士
語文別 英文
論文頁數 73頁
口試委員 口試委員-林秀娟
指導教授-謝淑珠
口試委員-傅子芳
口試委員-黃暉升
口試委員-何元順
中文關鍵字 檸檬素  精胺丁二酸合成酵素  N-乙醯半胱胺酸  瓜胺酸血症 
英文關鍵字 Citrin  argininosuccinate synthetase  N-acetylcysteine  citrullinemia 
學科別分類
中文摘要 檸檬素為一種位在肝臟粒線體內膜的載體蛋白,主要的功能為交換細胞質中的穀胺酸以及粒線體中的天門冬胺酸;運送到細胞質的天門冬胺酸再經由精胺丁二酸合成酵素(argininosuccinate synthetase, ASS)催化和瓜胺酸產生精胺丁二酸。之前的研究指出,缺乏檸檬素的病人之血液氧化壓力增加,細胞質nicotinamide adenine dinucleotide (NADH)堆積以及肝臟ASS缺乏等現象。由於檸檬素的缺乏會導致運送到細胞質的天門冬胺酸減少,進而影響到蘋果酸-天門冬胺酸路徑(malate-aspartate shuttle)的作用,造成細胞質內NADH的累積;另一方面,檸檬素缺乏致成的ASS缺乏之機制目前尚不清楚。因此本研究的目的是建立液相層析串聯質譜儀(liquid chromatography-tandem mass spectrometry)檢測細胞質NAD+與 NADH,以及細胞內榖胱甘肽(glutathione, GSH)與氧化態榖胱甘肽(oxidized glutathione, GSSG)之方法,並探討檸檬素缺乏引發ASS缺乏的作用機制,是否給予抗氧化劑甘胺酸或N-乙醯半胱胺酸(N-acetylcysteine, NAC)可以回復檸檬素缺乏所引發的傷害。研究中使用Huh-7細胞株,經由Menadione(提供超氧自由基)處理或電穿孔法將檸檬素基因的shRNA抑制檸檬素的表現後,觀察細胞ASS的活性與蛋白質表現情形,氧化壓力指標以及細胞凋亡程度。藉由流式細胞儀計算細胞Annexin V以及propidium iodide的表現來評估細胞凋亡程度;氧化壓力指標則藉由分析細胞質NAD+與NADH以及細胞內GSH與GSSG的變化測定;而蛋白質表現則以西方點墨法來檢測。研究中建立之方法可以測量5至5000 ng/mL的GSH及GSSG濃度;以及0.005到0.5 μmol/L之NAD+和NADH。兩種方法之不精密度(CV%)皆小於6%。在給予細胞超氧自由基的刺激會大幅降低細胞內GSH/GSSG比值(正常細胞138.7±24.3 vs. 超氧自由基1.56±1.01, p<0.01)、細胞質NAD/NADH比值(2.67±0.42 vs. 0.67±0.32, p<0.01)、以及細胞質ASS的表現(0.24±0.02 vs. 0.13±0.01, p<0.01)和活性(0.49±0.05 vs. 0.10±0.02, p<0.05)。細胞檸檬素表現被抑制後,GSH/GSSG(41.17±6.99 vs. 26.01±6.89)、NAD+/NADH(6.94±0.68vs. 4.75±0.57, p<0.05)以及ASS(0.12±0.02 vs. 0.05±0.001, p<0.05)的表現也明顯下降,而且細胞凋亡增加。給予甘胺酸或NAC皆可以有效降低細胞凋亡程度,並且使細胞存活率上升。在檸檬素抑制表現之細胞只有給予NAC才可以有效的改善細胞的GSH/GSSG與NAD+/NADH比值以及提升ASS之表現。甚至,在檸檬素抑制細胞合併給予超氧自由基的刺激下,NAC依然可以有效的使細胞氧化壓力下降,並且回升ASS的表現及活性。總結來說,經由氧化壓力的上升可能是檸檬素缺乏致成ASS缺乏之原因,而NAC則可有效降低氧化壓力,並回升ASS之表現。NAC可能可以做為檸檬素缺乏的新治療策略。
英文摘要 Citrin is a liver-type transporter for cytosolic glutamate and mitochondrial aspartate. The transported aspartate and cytosolic citrulline are converted into argininosuccinate through the catalysis of argininosuccinate synthetase (ASS). Patients with citrin deficiency showed increased oxidative stress, and decreased cytosolic nicotinamide adenine dinucleotide (NAD+) to NADH ratio, in accompany with reduced liver ASS activity. However, the mechanism of citrin deficiency-induced ASS downregulation is still unknown. Thus, the aim of this study is to establish liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for cytosolic NAD+/NADH and cell lysate GSH/GSSG measurement, and to investigate the effects of chemically or genetically-reduced ASS expression. And further to examine whether glycine or N-acetylcysteine (NAC) treatment would restore the changes. Huh-7 cells treated with menadione or citrin siRNA were analyzed for ASS protein expression and enzyme activity by western blotting and LC-MS/MS, respectively. Redox status was examined by cellular GSH/GSSG and NAD+/NADH ratio. We measured cells labeled by both Annexin V and propidium iodide, presenting apoptotic cells using flow cytometry. The LC-MS/MS methods showed the linear range from 5 to 5000 ng/mL for GSH and GSSG, 0.005 to 5 μmol/L for NAD+ and NADH, respectively. Imprecision (CV%) for these two methods were <6%. Menadione-treated cells exhibited a higher apoptosis rate and a decrease in cell viability. Moreover, menadione-induced oxidative stress resulted in depleted GSH (138.7±24.3 vs. 1.56±1.01, p<0.01), decreased cytosolic NAD+ (2.67±0.42 vs. 0.67±0.32, p<0.01), lower ASS expression (0.24±0.02 vs. 0.13±0.01, p<0.01) and activity (0.49±0.05 vs. 0.10±0.02, p<0.05). Similar to menadione-treated cells, citrin-knockdown cells showed lower ASS expression (0.12±0.02 vs. 0.05±0.001, p<0.05), GSH (41.17±6.99 vs. 26.01±6.89) and cytosolic NAD+ level (6.94±0.68vs. 4.75±0.57, p<0.05). Glycine or NAC pretreatment protected cells from oxidative stress-induced apoptosis and increased cell viability. However, only NAC administration prior to menadione-treatment or citrin-knockdown cells would restore the changes in GSH, NAD+ levels and ASS expression. Moreover, NAC supplement rescued these alterations caused by citrin-knockdown combined with menadione-treated cells, indicating the potency of NAC protection. Taken together, our data suggest that down-regulation of ASS expression in citrin-deficiency cells might be through the induction of oxidative stress. NAC, being able to restore the ASS expression, GSH/GSSG and NAD+/NADH ratio, might serve as a therapeutic strategy for patients with citrin deficiency.
論文目次 Abstract (in Chinese)................................................................................. I
Abstract (in English)……………………………………………………... III
Acknowledgement……………………………………………………….. V
Index……………………………………………………………………… VII
Table list………………………………………………………………….. IX
Figure list……………………………………………………………….… X
Appendix list……………………………………………………………… XI
Introduction
Citrullinemia………………………………………………………… 1
Classical citrullinemia (CTLN1)……………………………………. 1
Type II citrullinemia (CTLN2)….…………………………………… 2
Citrin deficiency and oxidative stress……………………………….. 4
Apoptosis and citrin deficiency …………………………………….. 5
Treatments for patients with citrin deficiency…..…………………… 5
Animal model for citrin deficiency……………………….………….. 7
Nitric oxide and argininosuccinate synthetase………………………. 8
Effects of glycine…………………………………………………….. 9
Effects of N-acetylcysteine…………………………………………… 10
Menadione……………………………………………………………. 11
Aims and strategies………………………………………………………... 13
Materials and methods
1. Cell culture……………………………………………………… 14
2. Citrin-knockdown cells…………………………………………. 14
3. Detection of apoptosis…………………………………………... 15
4. Cell viability determination…………………………………….. 16
5. Total protein analysis……………………………………………. 16
6. Measurement of GSH and GSSG………………………………. 17
7. Measurements of cytosolic NAD+ and NADH…………………. 18
8. Western blotting………………………………………………… 20
9. Measurement of ASS activity…………………………………... 21
10. Detection of superoxide………………………………………… 23
11. Statistical analysis………………………………………………. 23
Results……………………………………………………………………... 24
Discussion…………………………………………………………………. 29
Conclusion………………………………………………………………… 33
References………………………………………………………………… 34
Tables……………………………………………………………………… 48
Figures…………………………………………………………………….. 49
Appendixes………………………………………………………………... 64
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