鋅是生物體中重要的元素之一因為它扮演著許多蛋白質所需的催化及結構上輔助因子。鋅的缺乏是形成癌症的危險因子之一，因為它會導致生物體內的氧化壓力 (oxidative stress)、DNA 損害和基因突變。有學者在酵母菌的研究中發現鋅缺乏導致氧化壓力，其他在大鼠及細胞實驗中的研究亦指出鋅缺乏會提升氧化壓力。生物體在正常有氧代謝下會產生活性氧分子 (reactive oxygen species, ROS)，內生性抗氧化酵素可消除而不至於傷害生物體。若超過抗氧化酵素之負荷則會產生氧化壓力。許多研究指出氧化壓力之增加與心血管疾病有很大的關聯，因為氧化壓力增加，會導致內皮功能之異常而發生血管病變。然而許多實驗是在細胞層次或是病理下的成體動物中的研究，對胚胎血管發育影響的研究卻很少，主要是因為缺乏適當的實驗動物。此外，最近的研究指出在斑馬魚中鋅轉送子 LIV1 (zinc transporter LIV1, Zip6) 對於胚胎發育過程中 epithelial- mesenchymal transition (EMT) 階段是不可或缺的，其中涉及到信號傳導及轉錄激活蛋白3 (signal transducer and activator of transcription 3, STAT3)/Snail 訊息傳遞路徑。然而有關”鋅缺乏”對胚胎發育的分子機制探討卻很少，對血管發育的研究則是完全沒有。在研究中，我們以斑馬魚為模式生物探討鋅缺乏、氧化壓力與血管發育的關係。我們初步的研究顯示鋅缺乏會對血管發育造成影響，主要在斑馬魚區間血管及尾部靜脈血管叢部位，推測是鋅缺乏抑制血管內皮細胞之血管生成作用 (angiogenesis)。此外，血管專一基因的表現在鋅缺乏的胚胎中顯示較低量或異位的表現。另外，我們也發現胚胎在氧化壓力下血管發育也出現生長缺陷。最後，我們測試了鋅缺乏影響血管發育的分子機制是因為氧化壓力升高的關係。瞭解此機制，在新生兒預防醫學上可以避免因鋅代謝失調所造成的胎兒畸形或血管相關疾病。例如：腸病變性肢端皮膚炎 (acrodermatitis enteropathica )、嬰兒血管瘤 (infantile hemangioma) 等；也有助應用於抑制異常血管生成的相關疾病，如癌症。

Zinc is an essential nutrient for all organisms because it is required as a structural and catalytic cofactor by hundreds of proteins. Zinc deficiency is associated with increased levels of lipid and protein oxidation. In addition, the oxidative stress associated with zinc deficiency leads to increased levels of DNA damage. One previous study showed zinc deficiency can cause oxidative stress in yeast. Similar studies in animal and mammalian cells also showed that zinc limitation leads to increased oxidative stress. Many studies have been shown the connection between oxidative stress and vascular diseases. However, most studies have done in adult animals under pathological conditions, and very limited information about oxidative stress and vascular development during embryogenesis. Zinc is important in several biological processes including growth and development. Recent study showed zinc transporter LIV1 (Zip6) is important for Epithelial-mesenchymal transition (EMT) during the development mediated by STAT3/Snail signalings. However, it has limited knowledge regarding to the molecular mechanism on the impact of zinc deficiency in the developing embryo due to a lack of suitable experimental models, and no report has been shown the effect of zinc deficiency in vascular development yet.
Our preliminary data showed that zinc deficiency results in the defect in vascular development at intersegmental vessels (ISVs) and caudal vein plexus (CVP). In addition, vascular specific markers showed reduced or ectopic expression, suggested the impacts of zinc deficiency in vascular development. Understanding the effects of zinc deficiency in the developmental process will help us to prevent the prenatal zinc-related symptoms that might cause severe developmental malformation, such as acrodermatitis enteropathica (AE), infantile hemangioma. In addition, zinc depletion might serve as a new therapy for anti-angiogenesis related disease, such as cancer.

目錄
論文審定書..................................................................................................................... i
摘要................................................................................................................................ ii
Abstract ........................................................................................................................ iii
圖目錄........................................................................................................................... vi
表次及附件.................................................................................................................. vii
第壹章 前言................................................................................................................ 1
1.1 心血管疾病 .................................................................................................................... 1
1.2 鋅的重要性 .................................................................................................................... 5
1.3 斑馬魚的血管發育 ........................................................................................................ 7
1.4 利用斑馬魚為模式生物研究血管生成 ...................................................................... 10
第貳章 材料和方法.................................................................................................... 19
2.1 斑馬魚的繁殖，胚胎收集與培養 .............................................................................. 19
2.2 Morpholino 抑制基因表現 .......................................................................................... 19
2.3 顯微注射 (Morpholino microinjection) ...................................................................... 22
2.4 Total RNA萃取純化 ..................................................................................................... 22
2.5 cDNA 製作................................................................................................................... 23
2.6探針製作 ........................................................................................................................ 23
2.7原位組織染色(In situ hybridization) ............................................................................. 23
2.8 TUNEL Assay ............................................................................................................... 24
2.9 Quantitative PCR (Q-PCR) ........................................................................................... 25
2.10斑馬魚胚胎內ROS含量分析 ...................................................................................... 27
第參章 研究計畫目標與實驗流程圖........................................................................ 28
第肆章 實驗結果........................................................................................................ 30
4.1 鋅的特定螯合劑 TPEN 對斑馬魚的毒性 ................................................................ 30
4.2 TPEN 處理造成鋅缺乏會抑制斑馬魚的血管發育 ................................................... 30
4.3 TPEN 處理會降低或改變斑馬魚動靜脈基因 marker 的表現 ................................ 31
4.4 抑制 Zip4 基因表現造成鋅缺乏對斑馬魚的血管發育的影響 .............................. 31
4.5 抑制 Zip4 基因表現對斑馬魚的血管發育影響的專一性測試 .............................. 31
4.6 抑制 Zip4 基因表現 (Zip4 MO) 造成胚胎中鋅缺乏環境 ..................................... 32
v
4.7 鋅缺乏造成血管發育缺陷的分子機制 ...................................................................... 32
4.8 鋅缺乏造成氧化壓力進而導致血管缺陷 .................................................................. 33
4.8.1 氧化壓力導致斑馬魚 CVP 血管缺陷 ............................................................... 33
4.8.2 prdx1 knockdown 引起之氧化壓力會導致斑馬魚血管缺陷 ............................. 33
4.8.3 斑馬魚中鋅缺乏造成氧化壓力 ........................................................................... 34
5.1 鋅缺乏在血管生成和內皮細胞血管增生造成的影響 .............................................. 36
5.2 氧化壓力在血管和內皮細胞產生的影響 .................................................................. 36
5.3 鋅缺乏是引起氧化壓力的原因 .................................................................................. 36
5.4 鋅缺乏抗氧化基因和血管基因如何重建其基因表現的生物機制 .......................... 37
5.5 鋅在心血管疾病所扮演的角色 .................................................................................. 37
第陸章 結論................................................................................................................ 40
圖.................................................................................................................................. 41
Reference ..................................................................................................................... 60

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