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系統識別號 U0026-1808201716301000
論文名稱(中文) 探索葉酸缺乏所致魚鰾發育缺失及藥草萃取物調控胞外基質完整性的潛在性功能
論文名稱(英文) Exploring the mechanism underlying folate-deficiency induced swim bladder malformation and potential of herbal extracts modulating extracellular matrix (ECM) integrity
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 105
學期 2
出版年 106
研究生(中文) 游信瑄
研究生(英文) Hsin-Hsuan Yu
學號 T36044021
學位類別 碩士
語文別 英文
論文頁數 72頁
口試委員 指導教授-傅子芳
口試委員-張權發
口試委員-蔡振寧
中文關鍵字 葉酸  魚鰾  組織蛋白酶L  胞外基質  血管新生 
英文關鍵字 folate  swim bladder  cathepsin L  extracellular matrix (ECM)  angiogenesis 
學科別分類
中文摘要 葉酸是哺乳類懷孕期間的必須營養成分,包含貧血、神經管缺陷與癌症等許多疾病都與葉酸缺乏有關。在我們實驗室所建立的斑馬魚葉酸缺乏動物模式中發現斑馬魚魚鰾無法正常發育,而其組織蛋白酶L表現量會上升。這可能是造成魚鰾組織胞外基質受損而導致無法正常發育的原因。過去報導指出組織蛋白酶L會影響胞外基質完整性。而在胞外基質中蛋白酶的主要來源之一為吞噬細胞。在我們的研究中,我們探討吞噬細胞在葉酸缺乏誘導的魚鰾缺陷所扮演的角色,並且嘗試從藥草萃取物中找出可能具有干預胞外基質中蛋白酶的潛力藥物。我們使用兩種模式生物,包含血管螢光魚與葉酸缺乏的斑馬魚。主要是因為蛋白酶與抗蛋白酶的平衡會影響血管基底膜與胞外基質的完整性,而影響血管生成。我們發現,在葉酸缺乏的斑馬魚中,抑制巨噬細胞發育與抑制腫瘤壞死基因-α的活性皆會造成魚鰾無法正常發育。另外在藥草萃取物中發現,六神花、洋蓍草、桔梗、仙草和香椿,都會抑制血管發育。我們將非小細胞肺癌細胞異種移植至血管螢光魚中,並加入藥物觀察血管新生狀況。我們發現六神花與香椿在一天後都有抑制血管新生的現象。總結來說,我們發現巨噬細胞可能參與斑馬魚魚鰾發育。六神花萃取物可能具有影響血管新生的活性。這些天然藥草萃取物是否亦具有作為調控胞外基質完整性的潛能則需要進一步研究。
英文摘要 Folate is an essential nutrient for mammals during pregnancy. There are many disease, including anemia, neural tube defects and cancer correlating to the folate deficiency. Previously, we observed in the folate-deficient zebrafish larvae that increased activity of cathepsin L, a protease crucial for maintaining ECM integrity, has contributed the defective formation of swim bladder. In the current study, we attempt to confirm the role of inflammatory cells in the FD-induced loss of swim bladder and search from an herbal extracts library for the remedy with potential to intervene the ECM protease activity. Angiogenesis is an important process involving in physiological tissues development and pathological neoplasia. The activity and balance between protease and antiprotease are crucial for angiogenesis since they mediate the degradation of blood vessel basement and extracellular matrix (ECM), an essential step during angiogenesis. We used the transgenic fish with fluorescent vessels, Tg(fli1: EGFP), and the folate deficient larvae with defective swim bladder, Tg(zhsp70:EGFP-γGH), as our in vivo screening platforms. Our results showed that repressing macrophage development and inhibiting TNF-α activity aggravated the swim bladder malformation caused by folate deficiency. We found that fourteen out of the sixty-six extracts screened, including Spilanthes acmella (Linn) Murr, Achillea millefolium, Platycodon grandifloras, Mesona chinensis and Toona sinensis inhibited larval vessels development. The anti-angiogenesis activity of the selected extracts was further evaluated with the zebrafish tumor transplant model injected with non-small cell lung cancer H1299 cells. We found Spilanthes acmella (Linn) Murr and Toona sinensis could decreased angiogenesis on 1 dpi (day post injection) in xenograft model. In conclusion, our result suggested that phagocytes might involve in the development of swim bladder, and the Spilanthes acmella (Linn) Murr have the potential to regular the homeostasis of protease and modulate the ECM integrity.
論文目次 摘要 i
Abstract ii
Acknowledgement iii
Table of Content iv
List of Figure vi
List of Table vii
Abbreviations viii
I. Introduction 1
1. Folate 1
2. Zebrafish (Denio rerio) 1
3. Zebrafish folate deficiency model 2
4. Zebrafish swim bladder 3
5. Extracellular matrix (ECM) 4
6. Cathepsin L-1b 4
7. Neutrophil and macrophage in inflammatory lung disease 5
8. Angiogenesis and vasculogenesis 6
II. Rationale 8
III. Specific aims 9
IV. Materials and methods 10
1. Fish line and maintainance 10
2. Folate deficient zebrafish 10
3. Herbal extracts preparation and exposure 10
4. Western blot 11
5. Sudan black staining 11
6. Morpholino microinjection 12
7. RNA extraction 12
8. RT-PCR 12
9. Cell culture 13
10. Zebrafish xenograft assay 13
11. Statistic the morphological of blood vessel 13
12. Statistics analysis 14
V. Result 15
1. Increased expression of Cathepsin L in folate deficient larvae 15
2. Increased phagocytes in folate deficient larvae 15
3. Impact of knocking down neutrophils and macrophage to swim bladder development 15
4. Swim bladder maldevelopment in the FD larvae treated with macrophage inhibitor 16
5. Swim bladder maldevelopment in the FD larvae treated with the tumor necrosis factor alpha inhibitor 16
6. Down-regulated expression of protease mediating TNF-α release 17
7. Effects of herbal extracts on transgenic fish expressing fluorescence on blood vessel 17
8. Screen for herbal extracts affecting angiogenesis 18
9. Establish tumor metastasis zebrafish xenograft model 19
10. Screen for herbal extracts affecting angiogenesis in xenograft model 19
VI. Discussion 21
VII. Figure 25
VIII. Table 36
IX. Reference 40
X. Appendices 45
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