血管網絡的生長發育與建立需要有基因與訊息傳遞路徑的調控。對於血管發育的遺傳研究，斑馬魚是非常合適的脊椎生物模型。在先前的研究中，轉錄因子 islet2 和 coupTFIb 調控靜脈和區間血管 (intersegmental vessel, ISV) 及局部靜脈 (caudal vein plexus, CVP) 的生成。微陣列分析顯示，在 isl2 / coupTFIb MO 的調控下，一些 GTPase 相關蛋白基因表現量發生變化。GTPase 訊息傳遞路徑對於血管新生、細胞遷移、細胞增生等是重要的。在這次研究中，我們挑選了兩個新的 GTPase 相關蛋白基因 ect2 和 wasf1，調控斑馬魚的血管發育。原位組織染色結果顯示 ect2 和 wasf1 mRNA 在斑馬魚胚胎發育中的血管表現，顯示其對於脈管可能有影響。藉由 morpholino 分別降低 ect2 和 wasf1 的表現量造成 ISV 和 CVP 的缺陷，顯示 ect2 和 wasf1 會調控血管的發育及生長。為了確定是否因為細胞凋亡導致 ISV 和 CVP 的缺陷，我們進行了 TUNEL 檢測和 AO 染色。染色結果顯示血管缺陷不是由細胞凋亡引起的，可能是由於細胞增生和細胞遷移受抑制導致的。為了檢測分別注射 wasf1ATG MO 和 ect2e3i3 MO 造成之血管缺陷的分子機制，我們分析 wasf1ATG MO 和 ect2 e3i3 MO 與之血管 marker 的表現量。我們還顯示 GTPase 相關蛋白基因和 Notch 訊號之間的關係。為了了解 ect2 和 wasf1 對於 VEGF 和 BMP 訊號的關係，我們還利用西方墨點法進行偵測，發現大部分有下降的趨勢。總之，我們發現 GTPase 相關蛋白基因 wasf1 和 ect2 在斑馬魚胚胎血管發育中扮演重要角色。

Genetic programs and signaling pathways are required for proper growth and patterning of blood vessels. Zebrafish is a powerful vertebrate model organism to study genetic control vascular development. In our previous study, transcription factors islet2 and coupTFIb regulated vein and ISV (intersegmental vessel) formation. Microarray analysis showed that some GTPase related genes are changed in expression in isl2/coupTFIb MO. GTPase signaling has been shown important for angiogenesis, migration, proliferation etc. Here, we reported two novel GTPase related genes ect2 and wasf1 that control vascular development in zebrafish. In-situ hybridization shows that ect2 and wasf1 mRNA is expressed in developing vessels, suggested the roles in vasculature. Loss of ect2 and wasf1 by morpholino knockdown individually impairs the growth of ISV and CVP (caudal vein plexus), suggesting the role of ect2 and wasf1 in controlling ISV and CVP growth. To address whether the cell death contributes to the ISV and CVP defects in wasf1 MO and ect2 MO, we performed TUNEL assay and AO staining. The data showed that vascular defects do not caused by cell death, but likely due to the impairment of proliferation and migration. To test molecular mechanisms of vascular defects in wasf1 MO and ect2 MO, we examined the expression of vascular markers and we found the remodeling the expression of vascular markers in wasf1 MO and ect2 MO. We also revealed the relationship between those GTPase related genes and Notch signals. To test molecular mechanisms between VEGF signals, BMP signals and ect2, wasf1, we performed western blot. The data showed that VEGF and BMP signals is regulated by ect2 and wasf1. Together, we showed that GTPase related proteins wasf1 and ect2 play important roles for vascular development in zebrafish.

論文審定書 i
中文摘要 ii
Abstract iii
圖次 vi
表次 vii
縮寫 viii
壹、 前言 1
一、 斑馬魚作為模式生物 1
二、 斑馬魚血管發育 1
三、 VEGF-Notch 訊號影響斑馬魚動靜脈分化 2
四、 BMP 訊息傳遞路徑 3
五、 GTPase 訊息傳遞路徑 3
六、 Wasf1 3
七、 Ect2 4
八、 研究目的 4
貳、 材料與方法 6
一、 斑馬魚品系與飼養 6
二、 斑馬魚受精卵收集與培養 6
三、 嗎啉基/反義寡核甘酸 (Morpholino，MO) 6
四、 顯微注射法 (microinjection) 7
五、 RNA 萃取與 cDNA 製作 7
六、 探針 (probe) 製作 8
七、 原位組織染色 (in situ hybridization) 8
八、 TUNEL assay 10
九、 Acridine Orange (AO) staining 10
十、 聚合酶連鎖反應 (PCR) 10
十一、 High Fidelity PCR (HiFi PCR) 11
十二、 即時聚合酶連鎖反應 (Quantitative real time polymerase chain reaction, qPCR) 11
十三、 DNA電泳 12
十四、 DNA 純化 12
十五、 蛋白質萃取 (total protein extraction) 12
十六、 西方墨點法 (western blot) 13
十七、 影像拍攝 14
十八、 統計分析 14
參、 實驗結果 15
一、 實驗假說 15
二、 ect2 和 wasf1 的序列分析與基因表現 15
1. ect2 和 wasf1 在脊椎動物中具有高度的保守性 15
2. ect2 mRNA 和 wasf1 mRNA 於斑馬魚胚胎發育時的表現位置 16
三、 wasf1 對斑馬魚胚胎血管發育之影響 16
1. Knockdown wasf1 造成斑馬魚胚胎血管發育之缺失 17
2. Knockdown wasf1 造成斑馬魚胚胎血管缺失所衍生之現象 17
3. Knockdown wasf1 造成斑馬魚胚胎血管缺失的原因 18
4. knockdown wasf1 對於動靜脈基因 marker 表現量的調控 19
5. knockdown wasf1 對於其他組織器官的影響 19
四、 knockdown wasf1 會使 VEGF 和 BMP pathway 下游訊號下降 20
五、 ect2 對斑馬魚胚胎血管發育之影響 20
1. Knockdown ect2 造成斑馬魚胚胎血管缺失所衍生之現象 20
2. Knockdown ect2 造成斑馬魚胚胎血管缺失的原因 21
六、 Knockdown ect2 會使 VEGF signal pathway 下游訊號下降 22
肆、 問題與討論 23
一、 過度表現 wasf1 是否會導致血管過度生長 23
二、 為什麼 knockdown wasf1 使 qPCR 分析結果動靜脈基因 stabilin 表現量趨近不變 23
三、 wasf1 調控血管發育的分子機制 23
四、 Wasf1 與 GEF 是否為上下游關係 24
伍、 圖 25
陸、 表 41
柒、 參考文獻 43
捌、 附錄 47
附件一、其他 GTPase 相關蛋白基因在斑馬魚胚胎的表現位置 47
附件二、Knockdown ect2 影響動靜脈 marker 表現量 48
附件三、VEGF/NOTCH/BMP 訊息傳遞路徑調控 ect2 的表現量 49
附件四、藥品配置 50

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