血管發育對於脊椎動物正常生長極為重要。血管發育受到複雜的訊息路徑調控，但酵素調控蛋白質生物活性而影響訊息路徑的研究並不詳細。我利用斑馬魚作為模式生物探討基因如何調控血管生成。實驗室先前的研究中發現轉錄因子 Isl2/CoupTFIb 影響靜脈與體節間血管 (ISV) 生成。由微陣列分析結果中，指出 cpn1 可能受 Isl2/CoupTFIb 調控，推測 cpn1 會參與血管生成。 Carboxypeptidase N (CPN) 為血漿金屬蛋白酶，為兩個 CPN1 與兩個 CPN2 組成四聚體。然而目前尚未有研究指出 cpn1 與血管生成功能相關。比對 CPN1 胺基酸序列，發現在脊椎動物中保守性高，且 cpn1 作為 CPN 酵素活性中心，因此我們假設 cpn1 影響酵素活性調控血管發育。
原位組織染色顯示 cpn1 表現在側後中胚層、體節間血管、尾部靜脈叢與背部縱向血管，符合胚胎血管發育各時期，由此推測 cpn1 會直接影響脈管生成。利用注射 mRNA 或 Tol2 質體過度表現 cpn1，造成 ISV 與尾部靜脈叢 (CVP) 生長缺陷，與 isl2 morphant 表型相似。利用 TUNEL assay 與 Tg(kdrl:mCherry; fli:nEGFP)y7 魚種發現血管缺陷成因為內皮細胞增生與遷移受到影響，而非細胞凋亡造成。脈管相關基因表現發現靜脈基因 (mrc1, dab2) 表現量呈現下降的趨勢，這些結果顯示，cpn1 的過量表現，並不利於血管生長。可得知 cpn1 表現適量的重要性。
我感到意外的是當 knockdown cpn1 基因表現時也會導致 ISV 與 CVP 發育缺陷，由此可知 cpn1 對於血管發育有不可或缺的作用。利用第二種 morpholino 檢測 morpholino 對 cpn1 專一性與檢測 RT-PCR 中 cpn1 產物，確認 morpholino 的效率。我們同樣利用上述之 Tg(kdrl:mCherry; fli:nEGFP)y7 魚種發現 ISV 的細胞數與 ISV 生長都顯著減少，顯示內皮細胞增生與遷移功能受到影響導致血管缺陷。此外透過 TUNEL assay 發現有細胞的凋亡，但非在內皮細胞。我們進一步探討血管分子機制，檢測動靜脈基因 (flt4, mrc1, dab2, ephrinb2, gridlock, flk) 表現，皆有下降的趨勢。cpn1 的缺失造成血管發育的缺陷，由以上結果顯示 cpn1 對於血管發育過程中扮演不可或缺角色。
分析 cpn1 上游啟動子具 Isl2/CoupTFIb 結合位顯示 cpn1 可能受 Isl2/CoupTFIb 調控。利用 Luciferase assay 證實 cpn1 受 Isl2/CoupTFIb 的負向調控，與我們微陣列結果一致。我們也發現 cpn1 受 VEGF 與 Notch 訊息路徑調控而影響 ISV 的生長。cpn1 可能參與 BMP 訊息路徑，調控 CVP 的形成。
總結以上結果，我發現 cpn1 基因表現之精準調控對斑馬魚胚胎血管發育過程中，扮演重要的角色。

Vascular development is important for vertebrates and regulated by regulators via complicated signaling pathways. Enzymatic protein to control activation of signalings is critical but is not well understood. Zebrafish is a powerful vertebrate model organism to study genetic control vascular patterning. In our previous study, transcription factor Isl2/CoupTF1b regulated vein and ISV formation and genome-wide transcriptional analysis suggested that cpn1 is regulated by Isl2/CoupTFIb. Carboxypeptidase N (CPN) is a plasma zinc metalloprotease, which consists of two active subunits (CPN1) and two regulatory subunits (CPN2). However, no direct evidence shown the role of cpn1 in vascular function so far. Amino acid sequence alignment and phylogenetic analysis showed cpn1 is highly conserved in vertebrates. Thus, we hypothesize that cpn1 encode enzymatic subunits to perform CPN activity and function in vascular development.
In-situ hybridization showed cpn1 mRNA is expressed in developing vessels, sug-gested its function in vasculature. Overexpression of cpn1 impairs the growth of ISV (intersegmental vessel) and CVP (caudal vein plexus), which is similar to the phenotype of isl2 morphant. I showed that vascular defects is likely due to reduction of migration and proliferation. We further examined the expression of vascular markers and found that gain of cpn1 results in a decreased expression of vein/ISV specific markers, mrc1, flt4. These data indicates the important role of cpn1 in vascular development at optimal expression level.
Interestingly, knockdown of cpn1 by morpholino injection also causes vascular de-fects, suggesting the indispensable function of cpn1 in controlling ISV and CVP growth. We further showed the cpn1 morpholino knockdown works efficiently. TUNEL assay and AO staining showed that vascular defects do not caused by cell death, but due to the impairment of migration and proliferation by examining ISV growth and ISV cell numbers in Tg(kdrl:mCherry; fli:nEGFP)y7 fish. To test molecular mechanisms of vascu-lar defects in cpn1 morphants, I examined the expression of vascular markers. I found that loss of cpn1 results in a decreased expression of vein/ISV specific markers, flt4, mrc1, vascular markers flk, stabilin and artery markers ephrinb2. These data suggested that cpn1 is required for vascular development.
To examine the interaction between cpn1 and isl2/coupTFIb, we performed luciferase assay and showed cpn1 is regulated by isl2/coupTFIb, which is consistent with our mi-croarray results. We also revealed cpn1 is regulated by VEGF and Notch signalings.
Together, we showed cpn1 plays an important role for vascular development in zebrafish. We also uncovered a fine-tune regulation of cpn1 that controls vascular pat-terning mediated by Isl2-Notch-VEGF signaling to control ISV growth. In addition, cpn1 likely interacts with BMP signaling to function in CVP formation.

論文審定書 i
致謝 ii
中文摘要 iii
Abstract v
圖次 xi
表次 xii
Abbreviation xiii
壹、前言 1
血管生成的重要性 1
斑馬魚作為模式生物的優點 1
斑馬魚血管脈絡形成 2
動靜脈特化 2
VEGF 訊息路徑 3
Notch 訊息路徑對斑馬魚血管生成的調控機制 3
BMP 訊息路徑 4
cpn1 的篩選與研究動機 4
附圖 6
附圖一、血管脈絡形成 6
附圖二、動靜脈特化 7
附圖三、體節間血管生成 8
附圖四、VFGF訊息路徑 9
附圖五、Notch 訊息路徑 10
附圖六、BMP 訊息路徑 11
貳、材料與方法 12
一、斑馬魚之飼養與受精卵收集以及其培養 12
二、顯微注射 (microinjection) 12
三、Total RNA 萃取與 cDNA的製作 13
四、聚合酶連鎖反應 (PCR) 14
五、探針製作 14
六、原位組織染色 (In situ hybridation) 15
七、冷凍切片 16
八、細胞凋亡檢測 16
九、mRNA 合成 17
十、Tol2-cpn1 質體的構築 18
十一、影像拍攝 19
十二、Quantitative PCR (q-PCR) 19
十三、細胞培養 (Cell culture) 19
十四、pGL3-basic 載體建構選殖與 Luciferase assay 20
十五、轉染 (Transfection) 20
十六、西方墨點法 (Western Bolt) 21
十七、免疫共沉澱 (Co-Immunoprecipitation) 22
十八、斑馬魚蛋白質萃取 23
十九、統計分析 23
參、實驗結果 24
一、實驗假說 24
二、cpn1 序列分析其基因表現 24
1. cpn1 在脊椎動物中保有高度保守性 24
2. cpn1 mRNA 在斑馬魚胚胎各時期表現位置 25
三、cpn1 過度表現對血管發育的影響 26
1. cpn1 過度表現造成血管發育的缺陷 26
2. 過度表現 cpn1 與 isl2ATGMO 缺陷相似 27
3. 過度表現 cpn1 血管缺陷後的衍生的血液循環缺陷與心包膜腫大 (edema) 28
4. 過度表現 cpn1 所造成的缺陷由內皮細胞的遷移與增生功能受到抑制，非細胞凋亡造成 28
5. 過度表現 cpn1 造成靜脈基因 marker 表現量下降而造成血管缺陷 29
四、Knockdown cpn1 對血管發育的影響 30
1. Knockdown cpn1 造成血管發育的缺陷 30
2. Knockdown cpn1 血管缺陷後的衍生的血液循環缺陷與心包膜腫大 31
3. Knockdown cpn1 所造成的缺陷由內皮細胞的遷移與增生功能受到抑制，非細胞凋亡造成 31
4. cpn1e1i1 morpholino的專一性 32
5. knockdown cpn1 造成動靜脈基因 marker 表現量改變而造成血管缺陷 32
6. Protamine (Carboxypeptidase N抑制劑) 抑制脈管的生成 33
五、cpn1 基因表現受到精準調控的模型 33
六、 cpn1 的調控 34
1. cpn1 受到 VEGF/Notch 訊息路徑的調控。 34
2. 轉錄因子 Isl2 與 CoupTF1b 有交互作用 35
3. Isl2/CoupTF1b調控cpn1 36
七、總結: cpn1 影響血管發育機制 36
伍、問題與討論 37
一、cpn1 調控血管生長的分子機制 37
二、cpn1 與其受質 SDF-1 所影響功能或訊息路徑間的關聯性 37
三、CPN 活性的重要性 38
四、cpn1 平行或上游於 BMP 訊息路徑 39
五、cpn1 缺失導致血管性水腫 39
六、cpn1過度表現導致高血壓 40
陸、圖 41
柒、表 65
捌、參考文獻 67
玖、附錄 72
附件 72
附件一、pCSDest-cpn1 72
附件二、cpn1 Tol2CG2 73
附件三、pGL3-basic cpn1 promotor 0.5K (-454/-23) 74
附件四、pGL3-basic cpn1 promotor 1.2K (-1303/-23) 75
附件五、藥品配製 76
附件六、SDS-Polyacrylamide gel 81

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