氧化還原和遺傳訊息的平衡對於血管脈絡的生長是重要的。文獻指出，很多抗氧化基因(如sod1、grx2、prdx1和prdx2)對血管的生長是重要的。然而，目前大部分的研究都是在成體的病理狀態下；而關於氧化壓力在胚胎發育過程中對血管生成的影響研究卻是有限的。我在這論文中發現一個prdx1的新功能，prdx1在斑馬魚胚胎發育過程中對於血管的生長是扮演重要的角色。
Prdx1細胞質硫氧還原蛋白過氧化酶，可清除過多的過氧化氫和有機過氧化物。 我們首先藉由胺基酸序列比對和系統演化分析，發現PRDX1胺基酸序列從酵母菌到哺乳類動物都具有很高的演化保守性。我們藉由全覆式原位組織染色，得知prdx1的基因表現是在發育中的血管﹔藉由prdx1 morpholino的注射，發現區間血管和尾部靜脈血管叢的生長受到抑制，這些結果顯示prdx1可能是扮演促進區間血管和尾部靜脈血管叢生長的角色。為了測試prdx1 gene konckdown的效果是否專一，我們檢測splicing抑制效能且利用不同的prdx1 morpholino來阻斷splicing或是translation，得到相同的血管缺陷表型之結果。這顯示prdx1 morpholino具有高度專一性。我們也並探討造成血管缺陷的原因，發現prdx1 morphants的區間血管長度和細胞數都有減少的情形，顯示血管的缺陷可能是因為細胞增生和細胞遷移發生問題所造成，而不是血管內皮細胞凋亡所致。進一步探討血管缺陷之分子機制，我們檢測血管標誌物，發現prdx1的減少導致脈管的標誌物(ephrin-B2, flt4, mrc1, stabilin)表現量下降。顯示prdx1可能扮演調控血管發育的角色。
Prdx1缺少會導致血管的缺陷，顯示抗氧化功能對血管的發育是重要的，因此我們推論氧化壓力可能會造成血管生長的缺陷。胚胎藉由過氧化氫的處理。我們發現過氧化氫處理過的胚胎尾部靜脈血管叢的形成受到顯著影響，而區間血管的形成影響很小。此外，當經過prdx1 knockdown的胚胎再加上過氧化氫的處理雙重影響之下，造成更嚴重的血管缺陷表型，顯示氧化壓力是會影響血管生成的。此外藉由即時定量聚合酶連鎖反應的分析，進一步發現prdx1 knockdown會導致其他重要的抗氧化基因(sod1, sod2, catalase和prdx2)mRNA表現量下降。此外，我們並發現prdx1 konckdown會導致血球的標誌物gata1mRNA表現量上升，而血管內皮細胞標誌物flk1和fli的mRNA表現量減少。顯示其對血管血球母細胞分化有調控作用。
總結以上的結果，我們發現一個prdx1的新功能，即prdx1在斑馬魚胚胎發育過程中對於血管的生成扮演重要重要的角色。

Redox and genetic signaling homeostasis are required for proper growth and patterning of blood vessels. Many antioxidant genes, such as Superoxide dismutase 1 (sod1), Glutaredoxin 2 (grx2), Peroxiredoxin 1 (prdx1) and Peroxiredoxin 2 (prdx2) have been shown important for angiogenesis. However, most studies have done in adult animals under pathological conditions, and very limited information about oxidative stress and vascular development during embryogenesis. Here, we report a novel function of prdx1 that play critical roles in vascular growth during zebrafish development.
Prdx1 encodes a cytosolic thioredoxin-dependent peroxidase responsible for the remove of hydrogen peroxide and organic hydroperoxides. Amino acid sequence alignment and phylogenetic analysis of PRDX1 is highly conserved from yeast to mammalian. Our in situ hybridization results showed the expression pattern of prdx1 is in developing vessels. Knockdown of prdx1 by morpholino injection impairs the growth of intersegmental vessel (ISV) and cardinal vein plexus (CVP), suggesting the role of prdx1 in promoting ISV and CVP growth. To test the knockdown effect is prdx1 gene-specific, we examine the prdx1 morpholino knockdown efficiency and performed 2nd morpholino which block translational site of prdx1 results in similar vascular defects, suggesting the specificity of morpholino knockdown of prdx1. We further showed the reduction of ISV length and cell numbers in prdx1 morphants, indicating the vascular defects is due to a decrease of cell proliferation and migration, but not results from cell death in non-endothelial cells. To test molecular mechanisms of vascular defects that are associated with prdx1, we examined the expression of vascular markers. We found that a loss of prdx1 results in a decreased expression of vein/ ISV specific markers, flt4, mrc1, vascular markers stabilin and artery markers ephrin-B2. This indicates the regulatory role of prdx1 in controlling vascular development.
Loss of prdx1 results in vascular defects, suggest the antioxidant function is important. Thus, we test if oxidative stress could cause vascular defects in H2O2-treated embryos. Our data showed H2O2 treatment only impaired CVP formation but no obvious ISV defect. Interestingly, while H2O2-treated embryo combined with knockdown of prdx1, synergetic effects is observed, suggested the oxidative stress can disturb vascular development. In addition, knockdown of prdx1causes the reduced expression of main antioxidant genes (sod1, sod2, catalase and prdx2) by qPCR analysis. Moreover, we found the increased expression of blood marker gata1 coincident with the decreased expression of endothelial markers flk1 and fli in prdx1 MO, suggested prdx1 likely regulate hemangioblast fate decision. Together, we showed a novel function of prdx1 that play critical roles in vascular growth during zebrafish development.

論文審定書………………………………………………………………….………....i
中文摘要………………………………………………………………………….…...ii
Abstract ………………………………………………………………………….…...iv
圖表目錄…………………………………………….………………………………..ix
中英文名詞對照表…………………………………………………….…………...…x
Abbreviation………………………………………………………………….…….…xi
壹、 前言……………………………………………………………………..………..1
1.1氧化壓力………………………………………………………………………..1
1.1.1 ROS產生與傷害……………………………………………….………….. 2
1.1.2抗氧化的防禦機制…………………………………………………….…... 3
1.2 Peroxiredoxins (prdxs)家族…………………………………………………….3
1.2.1 Peroxiredoxin 1 (prdx1)……………………………………..………………4
1.2.2 Peroxiredoxin 2 (prdx2)…………………………………………..…………5
1.2.3 Prdxs的作用機制………………………………………………..……….…5
1.3斑馬魚血管的發育……………………………………………………….…….6
1.4 VEGF訊息傳導路徑…………………………………………………….…..…7
1.5 Notch訊息傳遞路徑在心血管發育過程中會調控細胞的二分化……………8
1.6 BMP訊息傳遞路徑會調控尾部靜脈血管叢(CVP)的特化…………………...9
1.7研究動機………………………………………………………………………10
1.8實驗架構………………………………………………………………………10
貳、 材料與方法……………………………………………………………………..11
2.1 實驗材料……………………………………………………………………...11
2.1.1斑馬魚模式生物………………………………………………………….11
2.1.2 Morpholino………………………………………………………….….…12
2.2 實驗方法……………………………………………………………….……..13
2.2.1斑馬魚的繁殖……………………………………………………….……13
2.2.2斑馬魚受精卵收集與培養…………………………………………….…13
2.2.3 顯微注射(Morpholino microinjection)…………………………………..13
2.2.4 Total RNA抽取…………………………………………………………...14
2.2.5 cDNA 製作…………………………………………………………….…15
2.2.6 切膠純化…………………………………………………………………15
2.2.7 探針製作…………………………………………………………………15
2.2.8 原位組織染色(In situ hybridation)………………………………………16
2.2.9 冷凍組織切片……………………………………………………………17
2.2.10 Acridine orange (AO) staining…………………………………………..18
2.2.11 TUNEL Assay……………………………………………………………18
2.2.12 Quantitative PCR (Q-PCR)……………………………………………...19
2.2.13 統計分析………………………………………………………….…….20
參、 實驗結果………………………………………………………………….….…21
3.1 PRDX1在脊椎動物中具有高度演化保守性…………………………..…….21
3.2 prdx1在斑馬魚胚胎發育過程中所表現的位置……………………….….…22
3.3 prdx1 knockdown對斑馬魚胚胎血管生成之影響………………….….….…23
3.4驗證prdx1 morpholino造成的血管缺陷…………………………….….……24
3.5檢測prdx1 knockdown所造成的phenotype是否專一………………...……25
3.6 prdx1 knockdown後血管缺陷衍生的表型………………………………...…26
3.7血管延遲生長是因為細胞凋亡或是細胞增生出錯所造成…………………27
3.8 prdx1 knockdown導致血管內皮細胞數的減少……………………………...28
3.9 prdx1 knockdown造成動靜脈marker表現量下降而造成血管缺陷…….…29
3.10氧化及還原壓力對斑馬魚胚胎血管生成的影響…………………………..30
3.11 prdx1 knockdown加上過氧化氫處理之加成試驗………………………….32
3.12 prdx1 knockdown對細胞內其他抗氧化酵素的影響………………………33
3.13 prdx1 knockdown導致血管內皮細胞表現量下降…………………………34
3.14 prdx1 knockdown對hemangioblast分化的影響……………………………35
肆、 問題與討論……………………………………………………………………36
4.1 prdx1與VEGF / Notch / BMP訊息傳遞的關係…………………………….36
4.2推測prdx1 knockdown造成血管的缺陷與VEGF的調控有關…………….37
4.3氧化反應和還原反應之間的恆定對血管生成的重要性……………………38
4.4 prdx1與氧化還原恆定的關係………………………………………………..39
4.5將胚胎prdx1 knockdown外加上氧化劑處理，所造成的加成作用………..40
4.6推測prdx1所造成的氧化還原失衡會破壞酪胺酸激酶受體(VEGFR2)
進而導致訊息傳導的失誤造成血管缺陷……………………………………41
4.7 Prdx1可能會促進癌症腫瘤的形成?................................................................42
4.8血管和神經的交互作用………………………………………………………43
伍、 結論……………………………………………………………………………44
陸、 Future work……………………………………………………………………..44
柒、 References………………………………………………………………………47
附錄…………………………………………………………………………………..68
藥品配置…………………………………………………………………………69
設備………………………………………………………………………………70
口試問題…………………………………………………………………………71

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