臺灣博碩士論文加值系統

葡萄糖六磷酸脫氫酶（G6PD）在五碳糖磷酸途徑(pentose phosphate pathway)中負責催化葡萄糖-6-磷酸(glucose-6-phosphate)轉換成6-磷酸葡萄糖酸內酯(6-phosphogluconolactone )，可產生還原態之菸鹼胺腺呤雙核酸磷酸(NADPH)，對許多生化反應提供還原能量維持細胞內抗氧化系統，保護細胞免受到氧化壓力的損害。臨床上針對酵素缺乏患者多研究與貧血相關，較少說明胚胎發育及早期的生長調控。因此，研究中利用斑馬魚動物模式，說明酵素在胚胎發育扮演的角色。在胚胎中注入反義核苷酸(Morpholino)能阻斷蛋白轉譯，減少斑馬魚G6PD活性，並發現胚胎發育產生遲緩現象。利用即時聚合酶連鎖反應（RT-PCR）偵測一連串受到上下游影響的氧化壓力相關基因。最後利用凋亡細胞的特性偵測活性含氧化物（ROS），及利用螢光染劑Acridine orange觀察細胞，此時當細胞受到G6PD活性下降影響，走向凋亡的細胞增加的趨勢。從實驗結果證明G6PD在胚胎發育扮演重要角色，能提供斑馬魚胚胎發育時所需的氧化還原物質，使胚胎能維持正常發育表現。

The Glucose-6-phosphate dehydrogenase (G6PD) is the housekeeping enzyme in pentose phosphate pathway, which the nicotinamide adenine dinucleotide phosphates (NADPH) is generated as intracellular reduction potentials to overcome the oxidative stresses in cells. To date, not much have been done to reveal the role of this important metabolic enzyme in early embryo development. In this study, we take the advantage of using zebrafish as an animal model to explore the role of G6PD in early embryo development. Through G6PD-morpholino (G6PD-MO) injecting, the G6PD gene expression is knockdown by blocking mRNA translation and subsequently the G6PD enzymatic activity is diminished. Through the phenotypic observations, retardation in embryo development in G6PD knockdown embryos was demonstrated. Using real-time PCR, a series of genes responsible for oxidative stress were measured to determine either up- or down-regulated in G6PD knockdown embryos. ROS measurement and acridine orange (AO) stain experiments have also shown that G6PD knockdown embryos have a higher tendency to undergo apoptosis. These data clearly suggested that G6PD is an important element for embryos’ normal developments, and possibly through its role in supplying reduction potentials for the needs during the embryo growing process.

Chinese Abstract…………….......……………………..................... i
English Abstract…………………..……………………………...... ii
Table of Contents…………………………….......…….................... iii
List of Figures…………………………………………................... v
List of Tables…………………………………................................. v
Chapter 1 Introduction.…………………………...…................... 1
1.1 The Pentose Phosphate Pathway…………………………..... 1
1.2 The Role of G6PD in PPP……………………….…….......... 2
1.3 G6PD Deficiency…………………………………………… 2
1.4 The Productions in PPP: NADPH…………………………... 4
1.5 Reactive Oxygen Species (ROS)……………………………. 4
1.6 ROS and disease and cell development……………………... 5
1.7 Antioxidant Systems: GSH, SOD, Catalase….……………... 6
1.7.1 Glutathione (GSH)………………………………………... 7
1.7.2 Superoxide dismutase (SOD)……………………………... 7
1.7.3 Catalase……………………………………………..…….. 8
1.8 Genes related to oxidative stress……………………………. 8
1.8.1 Cytoglobin (Cygb1 and 2)………………………………… 9
1.8.2 Dual oxidase 1 (Duox1)…………………………………... 9
1.8.3 nudix (nucleoside diphosphate linked moiety X)-type motif 1 (Nudt1)……………………………………………
10
1.8.4 NADPH oxidases 5 (NOX5)……………………………… 10
1.8.5 Forkhead box protein M1 (Foxm1)……………………….. 11
1.9 The possible role of G6PD in cell development…………….. 12
1.10 Motivation and experimental approach……………………. 12
Chapter 2 Materials and Methods….…………………………… 14
2.1 Zebrafish Maintenance………..……………………….......... 14
2.2 Microinjection of Morpholino Oligonucleotides...……......... 14
2.2.1 Morpholino Oligonucleotides……………………………... 14
2.2.2 Microinjection………………………………………..…… 15
2.3 RNA extraction and reverse transcription (RT)……………... 15
2.4 Quantitative real-time PCR…………………………………. 16
2.5 Protein extraction…………………………………………… 16
2.6 Western Blot Analysis………………………………………. 17
2.7G6PD Activity Determination……………………………….. 18
2.7.1 Sample solution preparation………………………………. 18
2.7.2 Calibration………………………………………………… 19
2.8 ROS Measurement………………………………………….. 20
2.9 Acridine Orange Staining……...……………………………. 20
2.10 Statistical analysis…………………………………………. 21
Chapter 3 Results............................................................................ 22
3.1 The Survival Rate of Zebrafish Embryos after Injections with Different Concentrations of G6PD Morpholino....22
3.2 The Effect of G6PD Knockdown in Embryonic
Development... 23
3.3 G6PD Knockdown Decreases G6PD Activity......... 23
3.4 G6PD Knockdown Could Induce Certain
Oxidative-Stress Related Genes Expression............. 24
3.5 ROS Measurement...................... 24
3.6 G6PD Knockdown Possibly Induce Cells Apoptosis.. 25
3.7 Oxidative Stress Possibly Induce Some other Antioxidant
System........................26
Chapter 4 Discussion........................................... 27
4.1 G6PD is the major source of NADPH to supply cells for
coping with oxidative stress attack................27
4.2 ROS accumulation triggers the apoptotic pathway in G6PD knockdown embryos............................27
4.3 Apoptotic cells accumulation resulted by G6PD
Knockdown........................................28
4.4 Apoptotic signaling pathway..................... 29
4.5 G6PD plays an important role in embryonic development..................... 30
References.............................. 39
Appendices............................... 51
List of Figures
Fig. 1 Survivals of Zebrafish embryos injected with
G6PD MO...........................................31
Fig. 2 The G6PD-MO injected Zebrafish embryos displayed abnormal phenotypes of somatotype and melanin pigmentation.......................32
Fig. 3 The G6PD-MO injected Zebrafish embryos displayed two different abnormal somatotype and melanin pigmentation, and that can be classified to two types.............33
Fig. 4 G6PD activities of Zebrafish embryos injected with different concentrations of G6PD-MO...............34
Fig. 5 G6PD knockdown and certain oxidative stress related gene expressions.................................35
Fig. 6 DCFH-DA stain to detect the ROS accumulation... 36
Fig. 7 G6PD knockdown induces cells apoptosis......... 37
Fig.8 Antioxidative genes were induced in G6PD knockdowns.38

List of Table
Table. Primers used in quantitative RT-PCR............ 49

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