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博碩士論文 etd-0723116-162647 詳細資訊
Title page for etd-0723116-162647
論文名稱
Title
探討受到isl2/nr2f1b正向調控之stap2b基因對於血管發育的作用
Functional characterization of isl2/nr2f1b activation target stap2b in vascular development
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
74
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2016-07-28
繳交日期
Date of Submission
2016-08-24
關鍵字
Keywords
血管生成、斑馬魚、尾部靜脈叢、區間血管、stap2b
vascular development, zebrafish, intersegmental vessel, stap2b, caudal vein plexus
統計
Statistics
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中文摘要
目前對於調控區間血管(Intersegmental Vessels, ISV) 與尾部靜脈叢(Cardinal Vein Pluses, CVP) 形成的相關基因以及訊息傳遞機制仍然未有充分的了解。我們實驗室先前發現轉錄因子Islet2 (isl2)與nr2f1b藉由Notch訊息傳遞路徑參與斑馬魚靜脈的分化以及區間血管的形成。為了近一步了解isl2/ nr2f1b的分子調控機制,我們利用微陣列分析篩選出可能受到isl2/ nr2f1b活化或是抑制的基因。在本研究中,我們將探討受到isl2/ nr2f1b活化的基因,經原位組織染色分析部分基因,發現在斑馬魚胚胎血管發育初期過程有所表現,因此推論這些基因可能與血管發育有關。
我分析其中一個受到活化的基因signal-transducing adaptor protein 2b (stap2b) 於血管發育過程中的功能性。stap2b為一調節蛋白 (adaptor protein) 在許多與免疫相關的細胞分子路徑中皆有其重要的功能性,然而並未有任何對於stap2b在血管發育功能性的相關描述。由原位組織染色的結果中得知stap2b於斑馬魚胚胎18體節階段至受精後48小時皆有表現,顯示出stap2b可能有參與血管的發育。當注射stap2b morpholio使其表現量下降後,我發現會造成區間血管以及尾部靜脈叢的缺失,推測stap2b對於血管的發育可能扮演重要的角色。近一步藉由測定stap2b morpholino的作用效率,以及利用stap2b mRNA回復因stap2b表現量下降所造成的血管缺失的型態,我確定stap2b morpholino的專一性。再者藉由AO染色與TUNEL分析的結果以及利用Tg (kdrl:mCherry; fli:neGFP y7)魚種檢視區間血管的細胞數,得知因為stap2b表現量下降所造成的血管缺失並非是由於細胞凋亡所致,但可能是由於血管內皮細胞的遷移以及/或者增生所造成。為了解stap2b詳細的分子作用機制,我檢視在注射stap2b morpholino後血管相關基因表現量的變化,並且發現血管相關基因的表現量於stap2b morpholino胚胎中有所下降。當stap2b表現量下降時會造成血管的缺失,而過度表現stap2b時也會促進區間血管的生長,表示stap2b對於血管的發育是需要的且具有促進的功能。
我接著探討stap2b與Notch訊息傳遞路徑以及BMP訊息傳遞路徑之間的交互作用,由結果發現Notch可藉由調控stap2b影響區間血管的生成,此外stap2b可能參與BMP訊息傳遞路徑而調控尾部靜脈叢的形成。
總括而言,microarray的結果指出許多isl2/nr2f1b下游基因可能與血管發育相關。stap2b在斑馬魚胚胎血管發育的過程中則也扮演重要的角色。
Abstract
Genetic and signal interaction that regulating the vein identity, intersegmental vessels (ISV) and cardinal vein pluses (CVP) patterning is still not fully understood. We previously identified the transcription factors Islet2 (Isl2) and nr2f1b required for specification of the vein and ISV growth mediated by Notch pathway in zebrafish. To understand the molecular mechanisms behind the isl2/ nr2f1b regulation, we performed the microarray experiments to identify the potential downstream targets. In this study, we focus on the potential activation targets and we confirmed several genes expressed in vessels during early development indicating those potential genes function in vascular development likely downstream of isl2/ nr2f1b.
We selectively assay the functional relevance of one potential target, signal-transducing adaptor protein 2b (stap2b). Stap2 is identified as an adaptor protein that has been showed functions in a variety of cellular signal pathways in immune system, however, there is no description of stap2b functioning in vascular development so far. Our in-situ hybridization results showed stap2b mRNA is expressed is expressed in developing vessels from 18S to 48 hpf stages, suggesting its roles in vasculization. Knockdown of stap2b by morpholino injection causes vascular defects, suggesting the role of stap2b in controlling ISV and CVP growth. We confirmed the specificity of stap2b MO knockdown by using 2nd MO interfere block splicing site, examining knockdown efficiency, and expressing stap2b mRNA to rescue stap2b morphants. AO staining and TUNEL assay showed that vascular defects do not caused by cell death, but due to the impairment of migration and/or proliferation by examining ISV integrity and ISV cell numbers in Tg(kdrl:mCherry; fli:neGFPy7) fish. To test molecular mechanisms of vascular defects in stap2b morphants, we examined the expression of vascular markers. We found that stap2b morphants showed a decreased expression of vascular markers flk and stabilin, consistent with the role of stap2b in vascular development. While loss of stap2b cause vascular defects, we expect overexpression of stap2b will enhance vascular growth. Overexpression of stap2b enhanced the fluorescent signals in vessels and the growth of ISV. These data suggest that stap2b is necessary and sufficient to promote vascular development.
We further examine the interaction between stap2b and Notch signals, we showed stap2b is regulated by Notch signals to control ISV growth. In addition, stap2b may interact with BMP signaling to function in CVP formation. Together, we conclude that our microarray results identify many vascular genes acting downstream of isl2/ nr2f1b pathway. We also showed that stap2b plays an important role for vascular development in zebrafish.
目次 Table of Contents
論文審定書………………………………………………………………………………i
致謝……………………………………………………………………………………...ii
中文摘要..........................................................................................................................iii
Abstract…………………………………………………………………………………v
圖次……………………………………………………………………………………...x
表次……………………………………………………………………………………..xi
Abbreviation…………………………………………………………………………..xii
壹、前言…………………………………………………………………………………1
一、背景…………………………………………………………………………….1
二、斑馬魚的血管發育…………………………………………………………….2
三、VEGF訊息傳遞路徑…………………………………………………………..3
四、NOTCH訊息傳遞路徑對於斑馬魚胚胎血管生成的調控………………….4
五、NOTCH訊息傳遞調控動靜脈的分化………………………………….........4
六、BMP訊息傳遞路徑……………………………………………………………5
七、nr2f1b與Islet1參與靜脈的分化…………………………………………….5
八、stap2b基因對於斑馬魚胚胎血管發育的影響……………………………….6
九、研究目的……………………………………………………………………….6
貳、材料與方法………………………………………………………………………….8
一、斑馬魚品系與飼養………………………………………………………….…8
二、斑馬魚受精卵收集與培養…………………………………………………….8
三、Mopholino的顯微注射 (Microinjection)…………………………………….9
四、RNA萃取與cDNA製作…………………………………………………..…9
五、DIG labeling RNA探針製作……………………………………………..…10
六、全覆式原位組織染色(Whole-mount In situ hybridization)…………..…10
七、冷凍切片……………………………………………………………………...11
八、Acridine orange (AO) staining……………………………………………….12
九、Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)……12
十、Tol2-stap2b與pCS-Dest-stap2b建構與注射…………………………….…13
十一、messenger RNA合成……………………………………………………...14
十二、即時定量PCR (Quantitative PCR)………………………………………..14
十三、影像拍攝…………………………………………………………………..15
十四、統計分析…………………………………………………………………..15
參、實驗結果…………………………………………………………………………..16
一、isl2/nr2flb正向調控之基因於斑馬魚胚胎中的表現……………………….16
二、stap2b於各物種間之胺基酸序列比對………………………………………16
三、stap2b於斑馬魚胚胎發育各時期表現位置……………………………......17
四、Knockdown stap2b對於斑馬魚胚胎血管發育的影響……………………..18
1. Knockdown stap2b造成斑馬魚胚胎血管發育的缺失………………..…18
2. Knockdown stap2b所致斑馬魚胚胎血管缺失衍生之現象……………..18
3. stap2b e2i2 morpholino的作用效率……………………………………….19
4.注射stap2bATG morpholino造成斑馬魚胚胎血管發育缺陷的原因……..19
5. stap2b對於血管相關基因marker表現量的調控………………..……...20
五、過度表現stap2b對於斑馬魚胚胎血管發育的影響…………………………21
1.過度表現stap2b促進區間血管的生長…………………………………...21
2.過度表現stap2b能促進內皮細胞之增生…………………………….…..22
3.過度表現stap2b能回復stap2b缺失之表現形態…………………….….22
六、stap2b與血管發育相關路徑的調控………………………………………..23
1. stap2b與相關路徑之間的調控…………………………………..………23
2. stap2b為Isl2/nr2fIb之下游基因………………………………….……...23
3. stap2b受到Notch訊息傳遞路徑之調控………………………………..24
4. stap2b受到BMP訊息傳遞路徑之調控…………………………………24
肆、問題與討論………………………………………………………………………..26
一、stap2b調控尾部靜脈叢發育之分子機制…………………………………..26
二、stap2b與JAK/STAT訊息傳遞路徑對於血管發育的調控………..……….26
三、stap2b與Notch訊息傳遞路徑間的調控…………………………………...27
四、stap2b於人類內皮細胞間的功能性相似……………………………...…...27
五、stap2b調控STAT3活性……………………………………………………..28
伍、圖………………………………………………………………………………...…29
陸、表…………………………………………………………………………………..47
柒、參考資料…………………………………………………………………………..49
捌、附錄………………………………………………………………………………..53
附件一、pCSDest-stap2b……………………………………………………..…..53
附件二、Tol2-fli-stap2b…………………………………………………………..54
附件三、注射stap2aATG morpholino對於胚胎血管發育並無影響…………….55
附件四、STAP2與JAK/STAT訊息傳遞路徑間的調控…………………….......56
附件五、藥品配置………………………………………………………………..57
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