肝癌是台灣癌症死亡排名的第二名，目前最常見的肝癌類型為肝細胞癌(Hepatocellular carcinoma, HCC)，肝細胞癌約佔原發性肝癌的90%。肝細胞癌的發生是多步驟、多因素協同作用的結果，正常肝臟歷經慢性發炎、脂肪肝、肝纖維化、肝硬化，肝細胞增生、最後發展成惡性腫瘤。蕾莎瓦（Nexavar，學名Sorafenib）是目前唯一被FDA所核准的標靶藥物，用來治療晚期肝細胞癌，可以抑制癌細胞成長且延長病人的壽命，但非適用全部病人，且其他用於治療肝癌的藥物成效不彰，因此迫切需要發展新的治療藥物。由於肝癌為富涵許多血管的腫瘤，且癌細胞的成長和轉移都和血管新生有密切的關係，所以抑制血管新生是治療肝癌的關鍵。Sorafenib 的其中一個功能就是抑制血管新生。
本論文始於利用血管螢光斑馬魚胚胎，篩選由國衛院生藥所謝興邦博士提供的326個小分子化合物，建立快速尋找抑制血管新生的藥物篩選平台。發現四個化合物 (LIB0256、LIB0307、LIB1F、LIB1O) 具有明顯抑制胚胎節間血管(ISVs) 的作用。另外，也測試了國衛院生藥所蔣維棠與徐祖安博士提供的兩個SRC抑制劑 (BPR1J419S1 (419S1)、BPR1J420S1 (420S1))，發現此二者具更強的抑制血管新生的作用。因此，選用此二者進一步進行抑制肝癌的實驗。
先前本實驗室己建立了HBx(p53-) 及src(p53-) 轉殖基因魚，分別在11及9個月會形成肝癌，且細胞周期相關基因表現明顯上升。利用11.3 個月大的HBx(p53-) 和8.7個月大的src(p53-)基因轉殖的成魚，眼窩注射兩個SRC抑制劑一週兩次，持續一個月後進行病理組織分析並抽RNA分析細胞周期相關基因之表現。發現419S1 和420S1及Sorafenib處理過的魚比起DMSO對照組，細胞周期相關基因(ccne1、cdk1、cdk2)表現量下降許多，同時H&E染色發現相較於DMSO對照組的呈現的肝癌病癥，419S1 和420S1及Sorafenib處理過的魚的肝組織呈現發炎、脂肪肝等肝癌前期病癥。
為了建立可更快速篩選抑制癌細胞增生和轉移的斑馬魚模式，我藉由異種移植方法，將大量表現Endothelin 1 (EDN1) 的293T細胞打入兩天大的斑馬魚胚胎之卵黃腔，測試419S1 和420S1抑制癌細胞增生和轉移的作用。發現419S1 和420S1可以有效的抑制癌細胞增生和轉移。
一般而言，小分子抗癌藥物因為毒性高，固然對癌細胞具有良好藥效，但卻因缺乏標靶性，而損害到正常細胞，引起副作用。因此，我也利用肝臟紅螢光的轉基因斑馬魚胚胎做肝毒性測試。發現420S1和 Sorafenib會減弱肝臟紅螢光強度以及縮小肝的大小，可能有肝毒性，而419S1與DMSO 對照組的結果相似，因此419S1對肝臟似乎較沒有毒性。
我的碩士論文建立了斑馬魚藥物篩選平台，證實了斑馬魚肝癌成魚或血管螢光斑馬魚胚胎、斑馬魚異種移植模式都可成為篩選抗肝癌的藥物之模式，並可利用肝臟紅螢光的轉基因斑馬魚胚胎檢查其肝毒性。相對於其他動物模型，斑馬魚可更快速地幫我們尋找新的抗癌藥物。

中文摘要 I
Abstract III
Table of contents V
Chapter 1 Introduction 1
1.1 General information of hepatocellular carcinoma (HCC) 1
1.1.1 Epidemiology 1
1.1.2 Risk factors 1
1.1.3 Preventions 2
1.1.4 Treatments 2
1.1.5 Other treatments 2
1.1.6 Targeted therapies 3
1.2 Zebrafish models 3
1.2.1 Cancer models 5
1.2.2 Liver cancer models 5
1.2.3 Zebrafish xenograft models 7
1.2.4 Angiogenesis 7
1.2.5 Zebrafish drug screening platform 8
1.3 Drugs for testing 10
1.3.1 326 small molecules and PTK787 10
1.3.2 BPR1J419S1, BPR1J420S1, and Sorafenib 11
Specific aim 12
Chapter 2 Materials and Methods 13
2.1 Transgenic zebrafish lines 13
2.2 Sources of compounds 13
2.3 Zebrafish maintenance 13
2.4 Embryo collection 14
2.5 Drug screening for angiogenesis inhibition 14
2.6 Titration of the compound concentration 15
2.7 Retro orbital injection (RO injection) 15
2.8 Liver tissue collection and paraffin section 15
2.9 Total RNA isolation 16
2.10 Reverse transcription-polymerase chain reaction (RT-PCR) 17
2.11 Quantitative polymerase chain reaction (Q-PCR) 18
2.12 Cell culture 19
2.13 Xenotransplantation assay to examine cell proliferation and migration in zebrafish 19
2.14 Hepatoxicity assay 20
2.15 Statistical analysis 21
Chapter 3 Results 22
3.1 Set up the drug screening platform 22
3.2 The identification of the small compounds with anti-angiogenesis effect using zebrafish larva 23
3.3 BPR1J419S1 and BPR1J420S1 exhibit strong anti-angiogenesis effect 25
3.4 BPR1J419S1 and BPR1J420S1 may prevent HCC formation in HBx(p53-) and src(p53-) transgenic zebrafish 25
3.5 BPR1J419S1 and BPR1J420S1 reduce tumor cell migration and proliferation in xenotransplantation model 26
3.6 The concentration of BPR1J419S1 shows less hepatotoxicity 28
Chapter 4 Discussion 29
Conclusion 33
Figures and Tables 34
Figure 1. VEGF signal pathway in angiogenesis and the three small molecules exhibit the anti-angiogenesis effect. 34
Figure 2. Structures of 326 small compounds. 36
Figure 3. Measurement of the lengths of ISVs. 37
Figure 4. Plate setting and the examples of anti-angiogenesis phenotype every screening. 38
Figure 5. Titrations of LIB1F, LIB1O, LIB0307, and their IC50 for anti-angiogenesis. 40
Figure 6. Titrations of 419S1 and 420S1, and their IC50 for anti-angiogenesis. 42
Figure 7. Adult zebrafish with HCC treated with 419S1 and 420S1 reversed HCC formation. 44
Figure 8. 419S1 and 420S1, and Sorafenib exhibit anti-proliferation and anti-migration abilities in xenotransplantation assay. 46
Figure 9. GFP-mCherry embryos treated with 419S1, 420S1, and Sorafenib and observe RFP intensity and liver size at 5 dpf. 47
Table 1 The primer sequence of Q-PCR for cell cycle-related genes ccne1, cdk1, and cdk2. 48
Supplementary Figures 49
Figure S1. Anti-angiogenesis screening for LIB1O and LIB1F derivatives. 49
Figure S2. HBx(p53-) and src(p53-) showed HCC at 11 and 9 months of stage. 53
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