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研究生： 李征原
論文名稱： 利用斑馬魚腦創傷模式探討神經血管重組
Zebrafish Model of Traumatic Brain Injury for Neurovascular Remodeling Research
指導教授： 莊永仁
口試委員: 林澤
劉意雯
學位類別： 碩士
Master
系所名稱： 生命科學暨醫學院 - 生物資訊與結構生物研究所
Institute of Bioinformatics and Structural Biology
論文出版年： 2012
畢業學年度： 100
語文別： 英文
論文頁數： 48
中文關鍵詞： 神經血管重組 、斑馬魚 、腦創傷
相關次數： 點閱：1  下載：0 
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    • 由中風或創傷所造成的腦損傷一直在世界各地都是主要的健康問題。除了高發生率與死亡率，腦損傷也導致大多數的患者長期知能與行動障礙。至今，腦損傷的治療方法都專注於神經保護，然而越來越多的證據顯示，在受損區域若能同時促進血管新生與神經再生，對病患的治療可有顯著的改善。透過這樣的臨床觀察，我們想知道是否增益神經血管修護環境，能有助於腦損傷的修復。
    為了瞭解神經血管修護環境在腦損傷後的修復機制，我們發展了一個腦損傷斑馬魚模式以探討小腦損傷後的神經血管再生。在細胞層次上，我們利用螢光標記觀察血管在小腦損傷後不同時間點的再生狀況。此外在分子層次上，我們利用微陣列分析在小腦損傷後，不同時間點基因表現的模式趨勢，以找出可能在神經血管修護環境激活的訊號傳遞路徑。最後，我們還嘗試利用一套多物體影像追踪與行為分析工具，計算斑馬魚的游泳距離、反向轉彎的次數及轉動的次數，來觀察斑馬魚小腦於泳動功能的修復。綜而言之，本研究成功利用轉殖基因斑馬魚，針對因意外事故造成的創傷性腦損傷，開發了一個低成本的腦損傷動物模式，藉以供研究者剖析在組織修復過程中，血管新生與神經再生對修復過程的影響。

    中文摘要. I Abstract.. II 致谢. . . . . III Abbreviations VII 1. Introduction 1 1.1 Traumatic brain injury and current treatment 1 1.2 Wound healing 2 1.3 Angiogenesis and Neurogenesis 3 1.4 Neurovascular niche 4 1.5 The advantages of zebrafish model in regeneration research 4 1.4 The objective of this study 5 2. Materials and Methods 6 2.1 Zebrafish line 6 2.2 Stab lesion assay 6 2.3 Bright field image capture 6 2.4 Immunohistochemistry staining 6 2.5 Vibratome 7 2.6 Confocal microscopy 7 2.7 Microarray 8 2.8 Behavior video tracking 8 3. Results. 9 3.1 Controlled injury created by needle stabbing in zebrafish cerebellum 9 3.1.1 Selection of fish 9 3.1.2 Selection of injury site 9 3.1.3 Design of needle device 9 3.2 Overall assessment of TBI zebrafish and the wound healing process 10 3.2.1 TBI assessment by appearance 10 3.2.2 Direct observation of the TBI brain 10 3.2.3 TBI effect on swimming pattern 11 3.3 Significant genes involved in angiogenic and neurogenic pathways were identified by time-series microarray analysis 11 3.3.1 Microarray data processing 11 3.3.2 Time-series gene expression profiling 11 3.3.3 Gene-enrichment pathway analysis 12 3.3.4 Comparison with previous TBI studies in the mouse and rat models 12 3.3.5 Core signaling pathways involved in zebrafish brain injury and recovery 13 3.4 Time lapse analysis on brain vasculatures remodeling and cell proliferation in the injury site 14 3.4.1 Visualization of zebrafish adult brain vasculatures 14 3.4.2 Angiogenesis and brain vasculature remodeling 14 3.4.3 PCNA signals was unevenly detected at the edge of neural stem cell niche near the injury site 15 3.4.4 Correlation between angiogenesis and PCNA positive cells 16 3.5 Behavior analysis of zebrafish after TBI 16 3.5.1 Behavior parameters affected by TBI 16 3.5.2 Determination on the zebrafish number in each behavior experiment 17 3.5.3 Preliminary data for behavior analysis under different environmental conditions 17 4. Discussion 20 4.1 Regrowth of blood vessels and accumulation of proliferating cells 20 4.2 Signalling pathway in neurovascular niche 22 4.3 The challenges in stabling zebrafish behaviour analysis model 23 List of Table 31 Table 1. RNA QA / QC information. 31 List of Figures 32 Fig 1. Schematic of stab lesion and the observation of cerebellum after stab lesion. 32 Fig 2. Regeneration of cerebellum after different time points post injury. 33 Fig 3. Heatmap of 1.5 fold upregulate and downregulate gene selection from microarray data. 34 Fig 4. Analysis of microarray data by STEM and PANTHER. 36 Fig 5. Recovery of blood vessel after Stab lesion. 37 Fig 6. Accumulation of proliferating cells after Stab lesion. 38 Fig 7. Interaction between blood vessel and proliferating cells during regeneration. 39 Supporting Information 40 Fig S1. Analysis of behavior parameters. 40 Fig S2. Analysis of behavior parameters with temperature-control. 41 Fig S3. Behavior set up for behavior analysis. 42 Fig S4. Design of syringe. 43 Fig S5. Track of Zebrafish behavior after TBI. 44 Fig S6. Lateral view of cerebellum blood vessels and PCNA positive proliferating cells during regeneration. 45 Fig S7. Comparison of vascular anatomy between embryonic and adult zebrafish. 46 Fig S8. 3D image of blood vessels and proliferating cells during regeneration. 47 Fig S9. Gene expression profiles of Klf4, Sox2 and Oct4 from microarray data. 48

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