過敏煎主要是由甘草、五味子、銀柴胡、防風、烏梅五種中藥材所組成，此藥物具有抗過敏及抗發炎的作用，其中甘草根據先前研究發現，甘草水萃物具有有效抗血管新生及抗腫瘤的活性，推測甘草與血管生成有密切的關係，而本篇論文主要研究目的為探討甘草萃取物是否具有影響血管增生之能力。透過斑馬魚Alkaline Phosphatase staining實驗，發現浸泡過敏煎及甘草後之斑馬魚胚胎的SIV branch points在(basket inside: 對照組 vs. 過敏煎, 1.00±0.18、2.00±0.30； basket outside: 對照組 vs. 過敏煎, 0.17±0.06、0.67±0.12)；(basket inside: 對照組 vs. 50 ppm甘草, 1.28±0.13、1.87±0.22； basket outside: 對照組 vs. 50 ppm甘草, 0.81±0.09、1.42±0.13)，皆有明顯促進血管增生的現象。接下來使用基因轉殖斑馬魚Tg(fli1:EGFP) 觀察血管增生的效果，發現在浸泡過敏煎後caudal vein plexus血管面積增加3.6%，也發現有促進血管增生的現象。最後在基因層次的研究發現， flt1、nrp1a (皆為血管內皮生長因子受體)、cdh5(細胞連接標記)基因表現量分別為對照組的 1.99倍、0.86倍及1.21倍。根據上述實驗結果，可得知過敏煎與甘草萃取物確實具有促進血管增生的能力。

Guo Min Jian (GMJ) is mainly composed of five kinds of Chinese medicine include Liquorice, Wu wei zi, Yin chai hu, Fang feng, and Wu mei. This Chinese medicine has anti-allergic and anti-inflammatory effects. According to previous paper, the water extract of Liquorice has anti-angiogenic and anti-tumor activity, it is inferred that Liquorice is closely related to angiogenesis. The main purpose of this thesis is to explore the pro-angiogenic ability of Liquorice extract. Results of Alkaline Phosphatase staining experiments, showed that the SIV of GMJ and Liquorice-treated zebrafish embryos had more branch points (basket inside: mock: 1.00±0.18 vs. GMJ-treated: 2.00±0.30；basket outside: mock: 0.17±0.06 vs. GMJ-treated: 0.67±0.12)；(basket inside: mock: 1.28±0.13 vs. 50 ppm Liquorice-treated: 1.87±0.22；basket outside: mock: 0.81±0.09 vs. 50 ppm Liquorice-treated: 1.42±0.13). Results of fluorescence recording of transgenic zebrafish Tg(fli1:EGFP) showed that GMJ-treated embryos displayed more broadly fluorescence expressions in the caudal vein plexus (the area of blood vessel increased by 3.6%) compared to no treatment mock group, indicating that both GMJ and Liquorice possess pro-angiogenic effects. Finally, from the genetic points of view, it was found that the expressions of flt1(VEGFR-1) and cdh5 (cell adhesion marker) genes in the GMJ-treated groups increased to 1.99 and 1.21 folds, but the expression of nrp1a (VEGFR) decreased to 0.86 folds. On the basis of these observations, we proposed that GMJ and Liquorice extract have pro-angiogenic effects.

謝誌......................................................I
中文摘要.................................................II
英文摘要................................................III
第一章 序論...............................................1
I.	甘草及甘草酸.......................................1
i.	介紹..............................................1
ii.	動物實驗..........................................3
II.	過敏煎............................................5
III.	血管新生..........................................6
IV.	斑馬魚的血管發育...................................8
V.	斑馬魚胚胎優勢....................................10
VI.     實驗動機.........................................10
第二章 材料與方法.........................................12
I.	斑馬魚(Danio rerio)..............................12
i.	飼養及維護.......................................12
ii.	胚胎收集.........................................12
II.	藥品製備.........................................13
i.	斑馬魚試驗.......................................13
a.	甘草(liquorice)..................................13
b.	甘草酸(Glycyrrhizin).............................13
c.	過敏煎(Guo Min Jian, GMJ)........................13
ii.	高效液相層析法....................................14
a.	甘草(liquorice)..................................14
b.	甘草酸(Glycyrrhizin).............................14
c.	過敏煎(GuoMinJian, GMJ)..........................14
III.	藥物浸泡.........................................15
IV.	RNA萃取(RNA extraction)..........................16
V.	反轉錄聚合酶連鎖反應(Reverse Transcription PCR,RT-PCR)....................................................16
VI.	即時聚合酶連鎖反應(Real-Time PCR, qPCR)...........17
VII.	鹼性磷酸酶染色Alkaline Phosphatase staining.......18
VIII.	雞胚絨毛膜試驗Chorioallantoic Membrane (CAM) Assay	........................................................19
IX.	螢光顯微鏡、照相系統及影像處理.....................20
X.	高效液相層析法（High Performance Liquid Chromatography, HPLC）...................................21
第三章 結果..............................................22
I.	證實過敏煎中含有甘草酸............................22
II.	觀察斑馬魚胚胎在浸泡不同濃度的藥物之存活率..........23
III.	觀察過敏煎對於Tg(fli1:EGFP)斑馬魚胚胎節間血管 (ISV)的影響.....................................................26
IV.	過敏煎對於斑馬魚胚胎腸下靜脈(SIV)血管發育的影響.....27
V.	甘草對於斑馬魚胚胎腸下靜脈(SIV)血管發育的影響.......28
VI.	甘草酸對於斑馬魚胚胎腸下靜脈(SIV)血管發育的影響.....30
VII.	過敏煎與甘草酸對於早期血管發育基因的影響............33
VIII.	甘草酸對於雞胚的血管發育影響.......................37
第四章 討論..............................................39
I.	過敏煎與甘草酸對於血管增生的效果及機制..............39
II.	甘草中影響血管發育的活性物質.......................40
III.	異甘草苷Isoliquiritin對於血管增生的影響............41
IV.	過敏煎各成分及甘草萃取物對於血管增生未來的發展.......42
第五章 參考資料...........................................44

圖表目錄
圖 一 過敏煎成分圖.........................................6
圖 二 斑馬魚藥物浸泡方法..................................15
圖 三 斑馬魚SIV branch points統計方式示意圖................19
圖 四 雞胚示意圖..........................................20
圖 五 過敏煎、甘草、甘草酸之HPLC分析.......................23
圖 六 過敏煎各濃度之存活率.................................24
圖 七 甘草各濃度之存活率..................................25
圖 八 甘草酸各濃度之存活率.................................25
圖 九 Tg(fli1:EGFP)斑馬魚ISV血管發育......................27
圖 十 過敏煎對於斑馬魚胚胎SIV的影響........................28
圖 十一 甘草對於斑馬魚胚胎SIV的影響........................30
圖 十二 甘草酸對於斑馬魚胚胎SIV的影響......................32
圖 十三 過敏煎對於flt1、cdh5以及nrp1a之qPCR分析............35
圖 十四 甘草酸對於flt1、cdh5、nrp1a、vegfaa以及kdrl之qPCR分析	........................................................36
圖 十五 甘草酸對於雞胚血管發育的影響........................38
圖 十六 甘草中的活性物質..................................41
表 一 qPCR 之引子序列.....................................18

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涂慶業(民108)。中藥之國老—甘草。藥師週刊， 2146~2147期