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研究生:王芊蕙
研究生(外文):Wang, Chian-Huei
論文名稱:THSD7AregulatesangiogenicpatterningviamediatingECprotrusions
論文名稱(外文):凝血酶敏感蛋白區域包含蛋白7藉由內皮細胞突出結構調控血管新生網絡
指導教授:莊永仁
指導教授(外文):Chuang, Yung-Jen
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物資訊與結構生物研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:英文
論文頁數:27
中文關鍵詞:血管新生
外文關鍵詞:angiogenesisvascularendothelial cell
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血管網絡的形成是一個被高度調控的過程。在此之中,包含了對血管內皮細胞移動、分支、和連接精密的調控。根據先前的文獻,內皮細胞正確的方向性移動和分支決定於正常的內皮細胞突出結構。然而方向性突出結構的相關機制仍未明確。在此篇文章中,我們提出一個新穎蛋白質—凝血酶敏感蛋白區域包含7A,簡稱THSD7A。這個新穎蛋白表現在班馬魚發育中的神經系統,而我們認為這個新穎蛋白具有調控內皮細胞突出結構的功能。
從我們的研究結果顯示,當我們利用morpholino抑制THSD7A蛋白質的表現時,受精30小時的班馬魚體節間血管的生長受到干擾。為了得到更多研究資料,我們利用共軛焦顯微鏡觀察THSD7A抑制的班馬魚,並檢察其體節間血的內皮細胞是否表現特殊形態和行為。結果顯示,當THSD7A被抑制時,血管內皮細胞被錯誤的引導並停留在細胞移動的初期,細胞突出結構廣泛的被延伸呈現扇形,因此導致體節間血管的生長被延遲。這個形態暗示內皮層細胞在縮回突出結構的機制上出了問題。除此之外,體節間血管上的內皮細胞也呈現不正常的分叉和錯誤連接的現像,使得受精後34小時班馬魚的血管網絡出現過度雜亂的圖形。
另一方面,一種在生物體外的細胞實驗—三度空間膠原蛋白的血管新生測試,也被運用來探討THSD7A的特殊功能。從此模式得到的研究結果顯示,THSD7A確實跟血管新生所產生的網絡圖形有關,包括調控內皮細胞的突出物結構和連接。統計資料也暗示我們THSD7A可能扮演了雙重性功能的角色,在不同濃度下,此蛋白對血管新生有不一樣的影響。
總而言之,這篇論文提供了THSD7A這個新穎蛋白影響血管新生的可能性,THSD7A可能藉由調控內皮細胞突出物,控制內皮細胞的分支和連接,進而影響血管新生的網絡圖形。

Vascular patterning is a highly organized process in angiogenesis. It involves precise control of endothelial cell (EC) migration, branching, and connection. Based on previous studies, directed EC migration and branching depends on correct protrusions of ECs. However, the underlying mechanism of directional EC protrusions is still unclear. Here we propose a novel secreted protein, THSD7A, which is expressed in the zebrafish developing neuronal system, contributes to EC protrusion regulation.
From our data, knockdown of THSD7A expression by injecting morpholino disrupted intersegmental vessel (ISV) sprouting of zebrafish at 30 hour-post-fertilization (hpf) stage. To acquire more information, we further examined the behavior and morphology of endothelial tip cells on the ISVs of Thsd7a-knockdown zebrafish by confocal microscopy. The data showed that morpholino knockdown of THSD7A slowed down ISV growth by misleading endothelia cells to stay longer in early stage of dynamic protrusion process, in which ECs extended numerous protrusions like a fan-shape. This phenotype implied malfunction of retracting excessive EC protrusions. Besides, ECs of ISVs formed ectopic EC branching and false connection, resulting in complex ISV pattern in 34hpf zebrafish.
On the other hand, an in vitro system, 3D collagen angiogenesis assay, was constructed for investigating the specific role of THSD7A. The data from in vitro 3D collagen angiogenesis assay proved that THSD7A was related to angiogenic patterning, including EC protrusions and connections. Statistic analysis also implied that THSD7A played a dual role in angiogenesis depending on its concentration.
Taken the in vivo and in vitro data together, this work suggests that THSD7A may mediate EC protrusion during angiogenic sprouting and plays an essential role in angiogenic patterning as a regular of EC branching and connection.

摘要 I
Abstract II
List of contents III
List of figures IV
1 Introduction 1
2 Materials and Methods 3
2.1 Fish stocks 3
2.2 Morpholino microinjection 3
2.3 Zebrafish THSD7A cloning and rescue 3
2.4 Real Time-quantitative PCR analysis 3
2.5 HUVEC isolation 4
2.6 Cell cultures of HUVEC, HEK 293 T 4
2.7 HEK 293 T expression system 5
2.8 3D collagen angiogenesis assay 5
2.9 ELISA (Enzyme-Linked Immunosorbent Assay) 6
3 Results and Discussions 8
3.1 THSD7A is required for ISV angiogenesis during zebrafish development 8
3.2 ISVs stayed in early step of migration process displaying protrusions without major direction in THSD7A-knockdown zebrafish at 29~34 hpf stage. 9
3.3 ISVs showed abnormal protrusions and connections across somite in THSD7A-knockdown zebrafish at 34 hpf stage. 10
3.4 Decreasing Flk1(VEGFR2) and increasing Flt4 (VEGFR3) expression in THSD7A-knockdown zebrafish suggested a possible genetic interaction. 11
3.5 Recombinant THSD7A mediated HUVEC angiogenic patterning in 3D collagen angiogenesis assay. 12
3.6 Recombinant THSD7A mediated EC protrusions in 3D collagen angiogenesis assay. 14
4 References 16

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