臺灣博碩士論文加值系統

循環系統發育過程中包含了血球生成及血管的形成，其中血球及血管皆源自於腹側中胚層 (ventral mesoderm) 。Etzf表現於ventral mesoderm、血球及血管發育位置上，透過morpholino抑制其表現後，ventral mesoderm中細胞增生的數量增多，指出Etzf對於ventral mesoderm形成重要。此外，抑制Etzf表現亦造成所有血球細胞markers表現量下降以及產生異常的血管網路。然而，上述Etzf之功能並不受BMP訊息傳遞路徑所調控。因此，透過我們的實驗數據顯示Etzf對於抑制ventral mesoderm的增生以及ventral mesoderm的分化扮演著重要的角色，且不透過BMP訊息傳遞路徑。Dtvx2同樣表現於發育中血球及血管位置上，當給予過量依賴Notch之持續活化型Dtvx2-ANK時，只有紅血球前驅細胞的marker gata1表現量上升，顯示Dtvx2能夠透過Notch訊息傳遞路徑來調控紅血球的分化。根據以上實驗結果，Etzf對於抑制ventral mesoderm增生及調控ventral mesoderm分化成血球或血管的過程為一個重要的因子，且不依賴BMP訊息傳遞路徑。而Dtvx2則可調控非典型Notch訊息傳遞路徑進而操控紅血球的分化。

Blood cells and blood vessels composing the circulatory system are both generated from ventral mesoderm. Although molecules that regulate blood cells and blood vessels formation have been well-characterized, however, those involved in specification of hemangioblastic fate in ventral mesoderm remained much less clear. Etzf, a transcription factor that expressed in the developing hematopoietic cells and vasculatures, and specifically expressed in ventral mesoderm at earlier stages. Morpholino knockdown of Etzf upregulated the proliferation of ventral mesodermal cells, suggesting Etzf is essential for formation of ventral mesoderm. Inhibition of the putative upstream regulatory pathway of etzf, BMP signaling, resulted in downregulation of ventral mesoderm marker eve1, but can not be rescued by etzf mRNA, suggesting etzf can be regulated by a signaling yet to be identified. Dtvx2 is specifically expressed in the axial vasculature and intersomitic vessels of the developing circulation system. Injection of Dtvx2-ANK only induced erythroid marker gata1, suggesting Dtvx2 is depended on Notch activation to positively regulate erythroid differentiation. Taken together, our data revealed Etzf is an essential factor for the inhibition of ventral mesodermal proliferation and blood cells and blood vessels differentiation, which is independent of BMP signaling.And Notch signaling may be regulated by non-canonical cytoplasmid regulators to operate erythroid differentiation.

目錄
介紹
1. 哺乳類循環系統發育與分子調控..............................1
2. 斑馬魚實驗動物模式之優點.................................4
3. 斑馬魚循環系統發育......................................5
3-1. 斑馬魚造血作用發育及分子調控............................5
3-2. 斑馬魚血管形成發育之分子調控與訊息路徑...................7
Etzf
4-1. Etzf轉錄因子與Ets轉錄家族...........................11
4-2. Etzf研究動機.......................................12
4-3. Etzf研究方法.......................................13
材料方法
5-1. 反股寡核酸 (Morpholino)..............................14
5-2. Deltex2 MO2功能確定.................................15
5-3. 體外 (in vitro) 合成Etzf/ Dtvx2 messenger RNA.......15
5-4. 回復實驗 (Rescue experiment)........................16
5-5. 構築deletion construct..............................16
5-6. DNA接合反應.........................................17
5-7. 質體轉型............................................17
5-8. 化學抑制劑浸泡斑馬魚胚胎..............................17
5-9. 全覆式原位雜交技術 (Whole-mount in situ hybridization)
....................................................18
5-9-1.胚胎復水 (Rehydration)..............................18
5-9-2.預雜交反應 (Prehybridization).....................18
5-9-3.雜交反應 (Hybridization)..........................18
5-9-4.雜交後胚胎之洗滌 (Post-hybridization wash).........18
5-9-5.抗DIG抗體 (Anti-Digoxigenin-AP) 吸附反應..........19
5-9-6.呈色反應..........................................19
5-10. 全覆式免疫染色 (Whole-mount immunohistochemistry)...20
結果
Etzf
6-1. 斑馬魚Etzf表現位置 (expression pattern) 分析...........21
6-2. Loss of function - MO抑制斑馬魚內生性Etzf對造血作用之影
響...................................................22
6-3. Etzf MO off target現象驗證............................25
6-4. Etzf MO抑制 - 專一性驗證..............................26
6-5. 超量表現去除Etzf功能性domain (deletion construct) 對造血
作用之影響............................................26
6-6. Gain of function – 超量表現Etzf mRNA對造血作用之影響...28
6-7. Ventral mesoderm增生情況分析..........................28
6-8. Loss of function - MO抑制內生性Etzf表現對血管之影響.....29
6-9. Etzf與訊息傳遞路徑 - BMP與MAPK訊息傳遞路徑..............30
討論
Etzf
7-1. Etzf在ventral mesoderm增生及分化能力中所扮演之角色......32
7-2. Etzf使細胞保持於ventral mesoderm細胞之狀態，造成
angiogenesis異常但不影響vasculogenesis................33
7-3. Etzf在hematopoietic lineage分化過程中之角色............34
7-4. Etzf之DNA binding domain與acidic domain所扮演之角色....34
7-5. Etzf參與造血作用之調控為必要或是必需之角色...............35
Dtvx2
8-1. Dtvx2家族............................................37
8-2. Dtvx2參與之訊息路徑...................................37
8-3. Dtvx2研究動機.........................................38
8-4. Dtvx2研究方法.........................................39
結果
Dtvx2
9-1. 斑馬魚Dtvx2表現位置分析................................41
9-2. Loss of function – Dtvx2 MO1抑制斑馬魚內生性Dtvx2表現對
造血作用之影響及回復實驗...............................42
9-3. Loss of function – Dtvx2 MO2抑制斑馬魚內生性Dtvx2表現.44
9-4. Gain of function – 超量表現Dtvx2-ANK mRNA對造血作用之
影響.................................................44
討論
Dtvx2
10-1. Dtvx2 MO解決方法.....................................46
參考文獻...................................................47
圖表......................................................52
圖表目錄
圖一、哺乳類造血作用位置之轉換...............................52
圖二、斑馬魚造血作用位置分佈.................................53
圖三、斑馬魚造血作用........................................54
圖四、斑馬魚血管生成及血管新生...............................55
圖五、血管形成之訊息路徑.....................................56
圖六、斑馬魚之Etz家族.......................................57
圖七、Etzf deletion construct設計..........................58
圖八、斑馬魚發育過程中Etv5 mRNA表現位置......................59
圖九、抑制內生性Etzf表現對斑馬魚胚胎型態之影響.................60
圖十、Etzf MO1、MO2抑制內生性Etv5表現及回復實驗...............61
圖十一、Etzf MO及p53 MO共同注射驗證MO之off-target現象........62
圖十二、去除Etzf DNA binding domain對造血作用之影響..........63
圖十三、去除Etzf acidic domain對造血作用之影響...............64
圖十四、超量表現體外合成之Etzf mRNA..........................65
圖十五、抑制內生性Etzf表現對ventral mesoderm之增生能力影響....66
圖十六、抑制內生性Etzf表現對Tg(fli1:eGFP) 斑馬魚胚胎血管形成
之影響.............................................67
圖十七、Etzf與BMP訊息傳遞路徑...............................68
圖十八、Etzf與MAPK訊息傳遞路徑..............................69
圖十九、典型Notch訊息傳遞路徑與非典型Notch訊息傳遞路徑.........70
圖二十、斑馬魚發育過程中Dtvx2 mRNA表現位置....................71
圖二十一、Dtvx2 MO1抑制內生性Dtvx2表現對rag1之影響及回復實驗...72
圖二十二、Dtvx2 MO1抑制內生性Dtvx2表現對pu.1之影響及回復實驗...73
圖二十三、Dtvx2 MO1抑制內生性Dtvx2表現對gata1之影響及回復實驗..74
圖二十四、Dtvx2 MO1抑制內生性Dtvx2表現對scl之影響及回復實驗....75
圖二十五、Dtvx2 MO1抑制內生性Dtvx2表現對gata2之影響及回復實驗..76
圖二十六、Dtvx2 MO1抑制內生性Dtvx2表現對mpo之影響及回復實驗....77
圖二十七、Dtvx2 MO2抑制內生性Dtvx2表現對scl、gata2、gata1、rag1
之影響...........................................78
圖二十八、RT-PCR驗證Dtvx2 MO2去除exon2......................79
圖二十九、超量表現Dtvx2-ANK mRNA............................80

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