血管的生長發育在胚胎發育過程中扮演重要角色，而利用斑馬魚作為模式生物可以方便探討基因如何調控血管生成。我們實驗室先前發現轉錄因子isl2/nr2f1b 調控血管發育；而利用微陣列分析出 Connexin 32 受到 isl2/nr2f1b 的調控。在脊椎動物中，由連接蛋白 (Connexin, cx) 所組成的間隙連接孔道來傳遞相鄰細胞之間的離子、小分子營養物質及訊息傳遞分子，以調節發育過程中細胞增殖、分化和遷移。cx32 的缺陷在一些病理症狀上有些異常現象的發生，文獻也指出 cx32 對小鼠受傷後血管的新生成有所影響，然而針對血管發育的研究卻是沒有的，此外，cx32 影響血管生成的訊息路徑到目前為止也尚未釐清。
比對 cx32 胺基酸序列，發現有兩個功能區在脊椎動物中保守性高。原位組織染色顯示其主要表現在頭部、初級血管環路的軀幹及尾部的部分，可見 cx32 對斑馬魚血管的生長發育可能有影響。注射 cx32 ATG morpholino 於斑馬魚胚胎，會造成胚胎血管生長上的缺陷，還有心包膜水腫和血液循環受阻的現象。TUNEL assay 與 AO staining 則顯示弱化 cx32 的表現會增加細胞凋亡，但位置可能不在血管內皮細胞處。藉由共軛交顯微鏡我們也觀察到斑馬魚區間血管中沒有絲狀偽足的產生，同時血管支數與細胞數的數量有明顯減少，說明弱化 cx32 會抑制細胞的移動和增生的能力。且動靜脈相關基因標記的表現量下降，但不會影響到神經、體節和心臟的基因標記表現。這些結果顯示，缺少 cx32 不利於血管生長。過度表現 cx32 不會影響胚胎血管發育，但可以回復 cx32MO 所造成的缺陷，顯示 cx32ATG morpholino 對於斑馬魚血管具有專一性。相關的訊息路徑探討方面，我認為缺少了 cx32 會降低 VEGF 和 BMP 訊息路徑。但 westen blot 結果顯示在 cx32 morphants 中 VEGF 和 BMP 訊息路徑相關蛋白表現量有很明顯的上升趨勢，推測可能是血管缺陷所導致的回饋補償作用。總結以上結果，發現 cx32 基因表現對斑馬魚胚胎血管發育過程中，扮演重要的角色。

The process of vascular development in vertebrates are mainly divided into vasculogenesis and angiogenesis. We previously identified the transcription factors Islet2 (isl2) and Nr2f1b were required for the growth of intersegmental vessels (ISV) and caudal vein plexus (CVP) in zebrafish. Microarray analysis showed Connexin 32 is regulated by Isl2/Nr2f1b. Gap junction protein Connexins32 (cx32) has been shown to function on the blood vessels in injured mice and to enhance angiogenesis in cell-based study, however, there is no study about the vascular development in vivo. In addition, signal pathways related to cx32 and angiogenesis have not been clarified. In this study, we first showed cx32 mRNA is expressed in developing vessels from 18S to 30hpf stages, suggesting its roles in zebrafish vascular development.
Knockdown of cx32 by morpholino injection caused vascular defects in ISV and CVP, suggesting the role of cx32 in vascular growth. TUNEL assay showed that vascular defects were not caused by cell death, but likely due to the impairment of endothelial cell proliferation and migration. Consistent with vascular growth defects, loss of cx32 affected the expression of the vascular markers flt4, mrc1, flk, stabilin, and ephrinb2. Furthermore, overexpression of cx32 did not impair the vascular growth, but could rescue the cx32 morphant, validating the specificity of morpholino knockdown. Finally, we examined the interaction between cx32 and multiple signals and observed that cx32 was regulated by VEGF signals for ISV growth, and BMP signals for CVP patterning.

目錄
論文審定書 i
中文摘要 ii
Abstract iii
圖次 vii
表次 viii
縮寫 ix
壹、前言 1
一、心血管系統 1
二、斑馬魚作為模式生物 1
三、斑馬魚血管發育 2
四、Connexin 3
五、VEGF signal pathway 3
六、BMP signal pathway 4
貳、實驗材料與方法 5
一、斑馬魚 5
二、顯微注射 (microinjection) 5
三、質體構築 6
四、plasmid 萃取 7
五、mRNA 合成 7
六、探針合成 8
七、原位組織雜交染色 (in situ hybridization) 9
八、冷凍切片 10
九、Total RNA 萃取 (Genemark RNA KIT) 10
十、cDNA 製備 (Roche cDNA KIT) 11
十一、聚合酶連鎖反應 (PCR) 11
十二、即時定量聚合酶連鎖反應 (q-PCR) 12
十三、DNA 電泳 12
十四、DNA 純化 13
十五、TUNEL assay 13
十六、AO (Acridine Orange) staining 13
十七、蛋白質萃取 14
十八、西方墨點法 (western blot) 14
十九、影像拍攝 15
二十、統計分析 15
參、實驗結果 16
一、研究動機和實驗假說 16
二、cx32 序列分析與基因表現 16
1.cx32 序列比對與功能區 16
2.cx32 mRNA 在斑馬魚胚胎發育過程中的表現位置 17
三、Knockdown cx32 對胚胎血管發育的影響 17
1.弱化 cx32 造成斑馬魚血管發育缺陷 17
2.弱化 cx32 血管缺陷後出現心包膜種大與血液循環缺陷 18
3.弱化 cx32 造成血管缺陷並非由細胞凋亡導致 19
4.弱化 cx32 造成細胞遷移受到抑制 19
5.弱化 cx32 造成細胞增生受到抑制 20
6.弱化 cx32 會降低動靜脈基因 marker 的表現量 20
7.弱化 cx32 不會影響胚胎其他器官發育 21
四、cx32ATG morpholino 專一性 21
五、過度表現 cx32 不會影響胚胎血管發育 22
六、弱化 cx32 會增加 VEGF 和 BMP 下游訊號的表現 23
肆、問題討論 24
一、cx32 調控血管發育的分子機制 24
二、cx32 與 actin, myosin 所影響之功能與訊息關聯性 24
三、cx32 與人類內皮細胞的功能相近 25
四、斑馬魚與人類 cx32 親緣關係之更新 25
伍、圖 27
陸、表 44
柒、參考文獻 47
附件一、脈管生成 (vasculogenesis) 及血管新生 (angiogenesis) 56
附件二、斑馬魚血管新生示意圖 57
附件三、血管內皮生長因子家族成員及其受體 58
附件四、BMP 訊息傳遞 59
附件五、connexin 結構 60
附件六、Pentabromopseudilin (PBP) 60
附件七、PBP 處理胚胎造成血管缺陷 61
附件八、弱化 cx32 與 PBP 的共抑制效果 62
附件九、TOPO-cx32 63
附件十、pDONR221-cx32 64
附件十一、pCSDest-cx32 65
附件十二、藥品配製 66
附件十三、SDS-Polyacrylamide gel 71

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