在血管的生長以及構築的過程中所必須仰賴的便是遺傳給予的調控訊息以及相關的訊息傳遞路徑。在研究遺傳訊息如何控制血管發育的過程，我們使用斑馬魚這種強而有力的模式生物來進行。在本實驗室先前的研究中已經證實轉錄因子Islet2和CoupTFIb可經由Notch訊號傳遞路徑調控靜脈血管構築以及區間血管的發育。經過DNA微陣列分析，我們發現有些GTPase相關蛋白基因在Isl2/CoupTFIb雙雙受到抑制的情況下，其表現量有改變的趨勢。GTPase相關基因在先前研究中被證實在血管新生、內皮細胞的遷移與增生等等血管發育的過程中扮演著不可或缺的角色。
在此我們針對一些GTPase相關蛋白基因進行研究：gtpbp1l、sept8b、ect2 和rhoub。我們認為這些GTPase相關蛋白基因能夠控制斑馬魚血管發育。首先我們利用斑馬魚的全覆式原位組織染色發現gtpbp1l、sept8b、ect2和rhoub的 mRNA皆表現在血管的相關位置之上，暗示了這些基因可能與血管發育有關。之後我們利用morpholino來分別抑制gtpbp1l、ect2和rhoub，並發現會造成區間血管以及尾部靜脈叢的缺失，我們猜測這些GTPase相關蛋白基因可以控制區間血管以及尾部靜脈叢的發育，另一方面我們與先前實驗相反利用過度表現基因的方法來測試GTPase相關基因在血管的調控，我們發現了過度表現sept8b和gtpbp1l會導致血管發育的缺失。
為了研究分別抑制gtpbp1l、ect2以及rhoub和過度表現sept8b造成血管發育缺失的分子機制，我們測試了在缺少或過多這些基因的情況下，血管相關標誌物的表現量。我們發現在gtpbp1l MO、ect2 MO以及rhoub MO和過度表現sept8b的情況下，血管標誌物因受到調控而改變表現量。隨後我們展示了這些GTPase相關基因與Notch、VEGF與BMP訊號傳遞路徑之間的關係，根據以上實驗結果我們認為GTPase相關蛋白基因gtpbp1l、ect2、sept8b和rhoub在斑馬魚血管發育的過程中扮演著重要的角色。

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 by notch signaling pathway. 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 several novel GTPase related genes gtpbp1, sept8b, ect2 and rhoub that control vascular development in zebrafish. In-situ hybridization shows that gtpbp1l, sept8b, ect2 and rhoub mRNA is expressed in developing vessels, suggested the roles in vasculature. Loss of gtpbp1l, ect2 and rhoub by morpholino knockdown individually impairs the growth of ISV and CVP (caudal vein plexus), suggesting the role of these GTPase related genes in controlling ISV and CVP growth. On the other hand, we used overexpression strategy to test the role of GTPase related genes in vasculature. We found overexpression of gtpbp1l and sept8b results in vascular defects.
To test molecular mechanisms of vascular defects in gtpbp1l MO, ect2 MO, rhoub MO and overexpressing sept8b embryos, we examined the expression of vascular markers and we found the remodeling the expression of vascular markers in gtpbp1l MO, ect2 MO rhoub MO and overexpressing sept8b embryos. We also revealed the relationship between those GTPase related genes and Notch, VEGF, BMP signals. Together, we showed that GTPase related proteins gtpbp1l, sept8b, rhoub and ect2 play important roles for vascular development in zebrafish.

論文審定書 i
致謝 ii
中文摘要 iii
Abstract v
目錄 vii
圖次 xi
表次 xii
縮寫表 xiii
壹、 前言 1
以斑馬魚作為模式生物研究血管發育 1
斑馬魚血管發育 1
VEGF-Notch訊號影響斑馬魚動靜脈分化 2
BMP訊息路徑 3
GTPase的篩選以及研究動機 3
貳、 材料與方法 5
一、 斑馬魚品系與飼養 5
二、 斑馬魚受精卵收集與培養 5
三、Mopholino的顯微注射 (Microinjection) 6
四、RNA萃取與cDNA製作 6
五、 DIG labeling RNA探針製作 7
六、 冷凍切片 8
1. 一次包埋 8
2. 二次包埋 8
3. 切片 8
七、 全覆式原位組織染色（Whole-mount In situ hybridization） 9
第一天 9
第二天 9
第三天 10
八、 Tol2-sept8b (gtpbp1l)與pCS-Dest-sept8b (gtpbp1l)建構與注射 10
1. BP reaction 10
2. LR reaction 11
九、 messenger RNA合成 11
十、 即時定量PCR (Real-time Quantitative PCR) 12
十一、影像拍攝 12
十二、統計分析 12
參、實驗結果 13
一、實驗假說 13
二、sept8b/gtpbp1l/ect2/rhoub 在脊椎動物中具有高度的保守性 13
三、GTPase相關蛋白基因mRNA在胚胎發育時期所表現之位置 14
四、knockdown GTPase相關蛋白基因對斑馬魚血管發育之影響 15
1. knowndown gtpbp1l 造成斑馬魚胚胎區間血管以及尾部靜脈叢缺陷 15
1.1 Knockdown gtpbp1改變血管marker表現量 15
2. knowndown ect2造成斑馬魚胚胎區間血管以及尾部靜脈叢缺陷 16
2.1 Knockdown ect2改變血管marker表現量 17
3. knowndown rhoub造成斑馬魚胚胎尾部靜脈叢缺陷 17
3.1 Knockdown rhoub改變血管marker表現量 18
五、過度表現GTPase相關蛋白基因對血管發育造成之影響 18
1. sept8b 過度表現造成斑馬魚胚胎尾部靜脈叢之缺陷 18
過度表現sept8b改變血管marker表現量 19
2 .gtpbp1l過度表現造成斑馬魚胚胎區間血管及尾部靜脈叢缺陷 19
2.1 gtpbp1l mRNA所造成缺陷可經由gtpbp1le4i4 MO挽救 20
六、GTPase相關蛋白基因受到上游訊息路徑調控 21
1. BMP訊號傳遞可以調控sept8b基因表現 21
2. gtpbp1l與上游訊息傳遞路徑Vegf、Notch與BMP之間的關係 21
3. ect2與上游訊息傳遞路徑Vegf、Notch與BMP之間的關係 22
4. rhoub與上游訊息傳遞路徑Vegf、Notch與BMP之間的關係 23
5. 結論 23
肆、問題與討論 24
1 為什麼只有knockdown gtpbp1l所造成之血管缺陷有觀察到心包膜腫大 24
2. BMP路徑是否經由GTPase相關蛋白基因影響血管生成 24
3. sept8bMO是否無效抑或是促進血管生長 25
4. Isl2/CoupTF1b如何調控GTPase相關蛋白基因之表現 25
5. gtpbp1l精準調控 (Fine-tune regulation) 斑馬魚血管發育 26
6. ect2及b之間是上下游或者平行關係 27
伍、圖 28
陸、表格 43
表1、ISH primer 43
表2、qPCR primer 43
表3、Morpholino 44
柒、附錄 45
捌、參考文獻 51

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