過去研究已知非氧化五糖磷酸途徑參與腫瘤發生，核糖-5-磷酸異構酶A（RPIA）是非氧化五糖磷酸途徑關鍵酵素之一。然而，在人類肝細胞癌中RPIA如何造成癌化尚不十分清楚。之前我們利用肝癌細胞株發現RPIA可經由降低PP2A來增加ERK磷酸化以促進細胞增殖及菌落形成。我們還利用RPIA過度表現的細胞株移植至裸鼠皮下的動物模式，證明過度表現RPIA可造成腫瘤的生長。這研究揭示人們對RPIA促成肝癌的新理解與提出RPIA作為治療肝癌的標靶的可能性。然而，過量表現RPIA造成肝癌的轉基因動物模型仍不存在。
斑馬魚已成為研究癌症和藥物開發的優秀模型。因此，在此論文中，我們使用肝特異性啟動子驅動RPIA表現在斑馬魚肝臟，利用此RPIA轉基因魚研究肝癌發生進程。使用F2的RPIA轉基因魚，我們收集了來自五個不同階段，3，5，7，9及11個月肝臟標本，利用QPCR分析脂肪生成因子，脂肪生成酶，纖維化標記基因和細胞週期的相關基因的表達。我們發現在RPIA轉基因魚5個月大時，脂肪生成因子，脂肪生成酶表現上升，同時使用HE染色及油紅染色檢測油滴，發現5個月時有脂肪肝形成。在RPIA轉基因魚7個月大時，纖維化標記基因表現上升，同時使用天狼星紅染色檢測纖維化，發現7個月時有肝纖維化。在RPIA轉基因魚9及11個月大時，細胞週期相關基因表現上升，同時使用HE染色檢測組織病理的變化，發現肝細胞增生和肝細胞癌。這些結果表明，在斑馬魚肝臟過度表達RPIA能促進肝癌的進展。
之前我們利用肝癌細胞株，已知過度表達RPIA能活化ERK信號傳導，在大腸癌細胞株中，過度表達RPIA能活化β連環蛋白途徑。在RPIA轉基因斑馬魚肝癌模型，我們使用免疫染色審查了ERK磷酸化和β-catenin蛋白量。我們發現在RPIA轉基因魚9及11個月大時，ERK磷酸化上升和β-catenin蛋白量也增加，並且在此階段PCNA增殖標記表現也上升。在將來我們希望利用RPIA轉基因斑馬魚肝癌模型篩選出治療手段，以防止RPIA介導的肝癌形成。

The non-oxidative pentose phosphate pathway is known involved in tumorigenesis. Yet the role of ribose-5-phosphate isomerase A (RPIA) in human hepatocellular carcinoma (HCC) is not well understood. Previously, we have discovered that RPIA regulates hepatocarcinogenesis via PP2A and ERK signaling in vitro using cell culture. We also demonstrated that RPIA regulates tumor growth in nude mice xenotransplant RPIA overexpressed cells. It sheds new light on RPIA as a potential biomarker and target for liver cancer therapy. However, the RPIA transgenic animal HCC model is still missing.
Zebrafish has become an excellent model for cancer study and drug discovery. Hence, we have generated RPIA transgenic fish using liver specific promoter drive human RPIA in zebrafish and examine the hepatocarcinogenesis at different stage. Using F2 of Tg (fabp10a: RPIA) transgenic fish, we collected the liver specimens from five different stage, 3, 5, 7, 9 and 11 months respectively, and analyzed the expression of lipogenic factor, lipogenic enzyme, fibrosis markers and cell cycle related genes using QPCR. The lipogenic factor and enzyme were up-regulated at 5 months, and using HE stain and Oil Red stain, and the liver has become fatty liver. At 7 months, we discovered the increasing expression of fibrosis markers, and using Sirius red staining, we found the hepatocyte has fibrosis. At 9 and 11 months, the cell cycle/proliferation markers were up-regulated; via HE staining we found the 9 to 11 months old fish showed hyperplasia and hepatocellular carcinoma. These results revealed that RPIA overexpression in zebrafish could promote liver cancer progression.
RPIA has been known to activate ERK signaling in liver cancer cell lines, and β-catenin pathway in colorectal cancer cell lines. We have examined the p-ERK and β-catenin by immunostaining, and found both p-ERK and β-catenin were activated at 9 and 11 months, and PCNA proliferation markers are also activated at this stage. In the future, we would like to use the RPIA transgenic fish liver cancer model to screen for therapeutic means for preventing RPIA mediated HCC formation.

誌謝 ............................................................................................................................... II
中文摘要 ...................................................................................................................... III
Abstract ........................................................................................................................IV
Table of contents ........................................................................................................... V
Chapter 1 Introduction ................................................................................................... 1
1.1 Hepatocellular carcinoma (HCC) .................................................................. 1
1.2 PPP pathway .................................................................................................. 2
1.3 ERK signaling ................................................................................................ 3
1.4 Zebrafish models .............................................................................................. 4
Chapter 2 Materials and Methods .................................................................................. 7
2.1 Transgenic zebrafish lines ................................................................................ 7
2.2 Zebrafish maintenance ..................................................................................... 8
2.3 Hematoxylin and eosin stain (HE stain) .......................................................... 8
2.4 Oil red staining ................................................................................................. 9
2.5 Sirius red staining ............................................................................................ 9
2.6 IHC staining ................................................................................................... 10
2.7 Liver tissue collection and paraffin section ................................................... 11
2.8 Total RNA isolation ....................................................................................... 11
2.9 Reverse transcription-polymerase chain reaction (RT-PCR) ......................... 12
2.10 Quantitative polymerase chain reaction (QPCR) ......................................... 13
2.12 Statistical analysis ........................................................................................ 14
Chapter 3 Results ......................................................................................................... 15
3.1 Expression of RPIA is dramatically increased in transgenic zebrafish compared to control zebrafish. ..................................................................... 15
3.2 Expression of lipogenic factors/enzyme were increased and accompanied with liver steatosis. ....................................................................................... 16
3.3 Expression of fibrosis markers were increased and liver fibrosis were obvious. ...................................................................................................................... 17
3.4 Expression of cell cycle/proliferation marker genes was obvious and accompanied with liver hyperplasia or HCC. .............................................. 17
3.5 PCNA is activated at 9 and 11 months and compared to HE stain with the same area. ..................................................................................................... 18
3.6 p-ERK is activated at 9 and 11 months of RPIA transgenic fish. .................. 19
3.7 β-catenin is activated at 9 and 11 months of RPIA transgenic fish. ............... 19
VI
Chapter 4 Discussion ................................................................................................... 21
Figures and Tables ....................................................................................................... 25
Fig. 1. Expression of RPIA is dramatically increased compared to control. ....... 25
Fig. 2. Expression of lipogenic factors and lipogenic enzyme were up at 5~7 months of RPIA transgenic fish. ............................................................... 26
Fig. 3. Oil red stain reveals the steatosis was developed at 5 and 7 months of RPIA transgenic fish. ................................................................................. 27
Fig. 4. Expression of fibrosis markers were obvious at 7 months of age. The QPCR analysis of fibrosis marker genes in RPIA transgenic fish. ........ 28
Fig. 5. Sirius Red stain reveals the fibrosis were developed at 7 months of RPIA transgenic fish. ............................................................................................. 29
Fig. 6. Expression of cell cycle/proliferation marker genes was obvious at 11 months of age. .............................................................................................. 30
Fig. 7. HE stain of all the stages’ hepatocyte and representative images from 9 and 11 months. ............................................................................................. 31
Fig. 8. PCNA immunostaining reveals the hyperplasia and HCC were developed at 9, 11 months of RPIA transgenic fish. ..................................................... 32
Fig. 9. p-ERK immunostaining reveals the phosphorylation of ERK were developed at 9 and 11 months of RPIA transgenic fish. .............................. 33
Fig. 10. β-catenin immunostaining reveals the β-catenin were enhanced at 9 and 11 months of RPIA transgenic fish. ............................................................. 34
Fig. 11. RPIA overexpressed in liver and colon cancer cell lines, and in transgenic fish caused p-ERK increase, -catenin increase and hepatocarcinogenesis. .................................................................................. 35
Fig. 12. Knockdown of RPIA in liver, colon and lung cancer cell lines. ............. 36
Table 1 The primer sequence of QPCR for lipogenic factors (pparg, srebp1, chrebp); lipogenic enzymes (dgat2, fasn, pap); fibrosis marker genes (col1a1a, ctgfa, hpse); cell cycle-related genes (ccne1, cdk1, cdk2). .......... 37
Supplementary Figure 1 Cloning of pTol2-fabp10a:RPIA; myl7: EGFP ............ 38
Appendix ...................................................................................................................... 40
Appendix 1. Pentose Phosphate Pathway ............................................................ 40
Appendix 2. Typical histological features of liver of HBx and src transgenic fish (B-H) in comparison with these of GFP-mCherry transgenic fish . 41
References .................................................................................................................... 43

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