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博碩士論文 etd-0727118-131141 詳細資訊
Title page for etd-0727118-131141
論文名稱
Title
參與膽固醇生合成的類NAD(P)H依賴性固醇脫氫酶(nsdhl) 對斑馬魚血管發育的重要性
NAD(P)H dependent steroid dehydrogenase-like (nsdhl) involved in cholesterol biosynthesis is critical for vascular development in zebrafish
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
66
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
2018-08-09
繳交日期
Date of Submission
2018-08-27
關鍵字
Keywords
Nsdhl、VEGF、BMP、血管新生、斑馬魚
Nsdhl, VEGF, BMP, Angiogenesis, zebrafish
統計
Statistics
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中文摘要
基因調控血管發育和血管網絡的生成在脊椎動物尚未完全了解。我們使用斑馬魚作為模式生物,發現轉錄因子 Islet2 (Isl2) 和 Nr2f1b 是區間血管 (Intersegmental vessel, ISV) 和尾部靜脈叢 (Caudal vein plexus, CVP) 生長所需的。我們進一步利用 microarray 發現NAD(P)H類固醇脫氫酶 (nsdhl) 基因可能受 Isl2/Nr2f1b 的調控而影響血管發育。 nsdhl 是參與膽固醇生合成中 lanosterol 的脫氫酶。過量的細胞膽固醇常常會導致異常增殖和遷移。另外,最近研究顯示膽固醇外流與血管生成控制有關。這裡,我們得知 nsdhl 在所有物種中具有高度保守性。 nsdhl mRNA 在血管內皮細胞表達,推測其在血管發育中扮演重要的作用。利用注射Morpholino (MO) 抑制 nsdhl 基因的表現,導致 ISV 和 CVP 發育缺陷,顯示 nsdhl 對血管發育有不可或缺的重要性。我們進一步檢測血管動靜脈基因 (flk, mrc1, stabilin, flt4和ephrinb2) 分子層次的表現,皆有下降的趨勢,符合血管發育的缺陷。為了證明細胞凋亡是否會造成 nsdhl MO 中 ISV 和 CVP 的缺陷,我們進行了 AO staining 和 TUNEL assay 測定。結果顯示,血管缺陷不是由細胞凋亡引起的,而可能是細胞增殖和遷移造成的損害。我們還發現了 nsdhl 與 Notch/VEGF 信號之間的關係。此外 nsdhl 可能參與 BMP 訊息調控路徑而調控尾部靜脈叢的形成。總結上述結果,我們證明 nsdhl 在斑馬魚血管發育中有重要的作用。
Abstract
Genetic conrol of vascular development and patterning in vertebrates is not fully understood. We used zebrafish as a model organism to identify transcription factors Islet2 (Isl2) and Nr2f1b required for the growth of intersegmental vessels (ISV) and caudal vein plexus (CVP). We further identify NAD(P)H steroid dehydrogenase-like (Nsdhl) gene is positively regulated by Isl2 /Nr2f1b. Nsdhl is a dehydrogenase that participates in sterol production in cholesterol biosynthesis. Excess cellular cholesterol often leads to abnormal proliferation and migration. In addition, recent study showed cholesterol efflux related to angiogenesis control.
Here, we show Nsdhl is highly conserved in all species. Nsdhl mRNA is expressed in vessels, suggesting its roles in vascular development. Knockdown of Nsdhl expression by Morpholino (MO) disrupts the growth of ISV and CVP, suggesting the role of Nsdhl in controlling ISV and CVP growth. Consist with the defects in vascular development, we examined the expression of vascular markers flk1, mrc1, stabilin, flt4 and ephrinb2 and we found the remodeling the expression of vascular markers in Nsdhl MO. To address whether the cell death contributes to the ISV and CVP defects in Nsdhl MO, we performed AO staining and TUNEL assay. The data showed that vascular defects do not caused by cell death, but likely due to the impairment of proliferation and migration. We also revealed the relationship between Nsdhl and Notch/VEGF signals. In addition, Nsdhl may interact with BMP signaling to function in CVP formation. Together, we showed that Nsdhl plays important role for vascular development in zebrafish.
目次 Table of Contents
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、血管生成的重要性+1
二、斑馬魚模式生物+1
三、斑馬魚血管脈絡+2
四、VEGF 訊息傳遞路徑+3
五、Notch 訊息傳遞路徑+4
六、BMP 訊息傳遞路徑+4
七、nsdhl基因篩選及研究動機+5
貳、實驗材料與方法+7
一、斑馬魚胚胎培養+7
二、Morpholino 的配製及顯微注射 (Microinjection)+7
三、Total RNA 萃取+8
四、cDNA製作 (Roche cDNA Kit)+8
五、聚合酶連鎖反應 (Polymerase chain reaction, PCR)+9
六、DNA電泳+9
七、DNA純化+9
八、DIG-labeling RNA探針製作+10
九、原位組織染色 (Whole-mount In situ hybridization)+11
十、TUNEL assay (Terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling assay)+12
十一、AO (Acridine Orange) staining+13
十二、即時聚合酶連鎖反應 ( Quantitative real time polymerase chain reaction, qPCR)+13
十三、影像拍攝+13
十四、統計分析+13
參、實驗結果+14
一、實驗假說+14
二、nsdhl 物種間胺基酸序列比對+14
三、nsdhl mRNA 於斑馬魚胚胎發育各時期表現位置+15
四、Knockdown nsdhl 對於斑馬魚血管發育的影響+15
1.Knockdown nsdhl 造成血管發育缺陷+15
2.Knockdown nsdhl 造成血管缺陷的其他影響+16
3. Knockdown nsdhl 所造成的缺陷原因+17
4. Knockdown nsdhl 對於血管相關基因 marker 表現量的影響+18
5. Knockdown nsdhl 對於胚胎器官發育的影響+18
五、 nsdhl和血管發育相關路徑的調控+18
1. nsdhl 受 VEGF/Notch 訊息路徑調控+18
2. nsdhl 受 BMP 訊息路徑調控+19
肆、問題討論+21
伍、圖+24
陸、表+34
柒、參考文獻+36
捌、附錄+43
附件一、脈管生成 (vasculogenesis)及血管新生 (angiogenesis)+43
附件二、斑馬魚脈管生成+44
附件三、斑馬魚血管新生+45
附件四、斑馬魚尾部靜脈叢+46
附件五、內皮生長因子-VEGF家族及其受體+47
附件六、Notch訊息傳遞路徑+48
附件七、VEGF及Notch訊息傳遞路徑影響動靜脈特化+49
附件八、BMP訊息傳遞路徑+50
附件九、Nsdhl基因參與膽固醇生合成 (Cholesterol synthesis)+51
附件十、藥品配製+52
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