臺灣博碩士論文加值系統

動脈粥樣硬化為動脈的慢性退化及動脈壁的逐漸硬化，是心血管疾病最常見的死因之一，缺血性心臟病、中風及週邊動脈阻塞等等都和粥狀動脈硬化有關，為了提前預防動脈硬化的發生，標靶顯影、治療早期血管硬化部位的技術日趨重要。研究顯示單核球會累積在許多動脈內的脂肪硬塊與斑塊，或是在其他受損的血管內皮細胞，因此單核球的標靶被視為早期動脈硬化診斷的工具。
單核球趨化蛋白-1 (MCP-1)具有誘導單核球趨化和激活單核細胞的雙重功能，在人體各器官的疾病中均有表達，並在患部吸引單核球的聚集與作用，促使單核球穿過內皮細胞進入血管壁，單核球也因此分化為巨噬細胞和泡沫細胞，形成動脈粥樣硬化。本實驗主要在研究氧化鐵奈米粒子，並在表面接有一段單核球趨化蛋白-1的氨基酸序列，利用此胺基酸序列讓氧化鐵奈米粒子對單核球有專一性鍵結，做為動脈粥樣硬化前期的顯影工具。
細胞實驗中利用不同種細胞測試可觀察到，接有MCP-1胺基酸序列的氧化鐵奈米粒子對單核球有專一性鍵結，而動物實驗中則使用apolipoprotein E (ApoE)基因缺陷小鼠，並餵食四周的高血脂食物以促進動脈粥樣硬化的產生作為實驗組，從小鼠尾靜脈注射氧化鐵奈米粒子，並利用magnetic resonance imaging (MRI)、in vivo imaging system (IVIS)顯影觀察氧化鐵奈米粒子的循環分布，發現在單核球聚集的器官或部位中，都有氧化鐵奈米粒子的聚集，尤其在主動脈的分布較其他組別明顯，最後利用油紅染動脈硬化組織和普魯士藍染氧化鐵奈米粒子，皆能證明接有MCP-1胺基酸序列的氧化鐵奈米粒子具有動脈粥樣硬化標定的能力。

Commonly, atherosclerosis is a multifactorial inflammatory disease that would progress silently for long period and widely accepted as the main cause of cardiovascular diseases. To prevent atherosclerotic plaques generating, imaging early molecular markers and quantifying the extent of disease progression are the effective ways in biomedical engneering. During the inflammation, circulating monocytes leave the bloodstream and migrate into incipient lipid accumulation in the artery wall, following conditioning by local growth factors and proinflammatory cytokines. Therefore, monocyte accumulation in the arterial wall can be observed in fatty streaks, rupture-prone plaques, and experimental atherosclerosis. Targeting monocytes as a strategy to diagnose atherosclerotic lesions could yield a molecular imaging tool that would detect the early-stage atherosclerosis, quantify the extent of plaque progression.
The objective of the work is to research monocyte-targeting iron oxide magnetic nanoparticles (MNPs), which are incorporated with the peptides derived from the chemokine receptor CCR2-binding motif of monocyte chemoattractant protein-1 (MCP-1) as diagnostic tools for potential atherosclerosis. In this study, MCP-1-motif MNPs had specific affinity to monocytes using in vitro fluorescence imaging. In addition, with MNPs injection in ApoE knock-out mice (ApoE KO mice), the well-characterized animal model of atherosclerosis, MNPs were found in specific organism or regions which had monocyte accumulation, especially the aorta of atherosclerosis model mice, through the in vivo imaging system (IVIS) imaging and magnetic resonance imaging (MRI). We also perform Oil Red O staining and Prussian Blue staining to confirm the specific affinity of MCP-1-motif MNPs.

誌謝 i
摘要 iii
ABSTRACT iv
CONTENTS vi
LIST OF FIGURES ix
LIST OF TABLES xii
Chapter 1 Introduction 1
1.1 Atherosclerosis 1
1.1.1 Chemokine 2
1.1.2 Monocyte chemoattractant protein-1 3
1.1.3 Targeting Strategy 4
1.2 Nanoparticles 5
1.2.1 Magnetic Nanoparticles 6
1.2.2 Core-Shell Structure 7
1.2.3 Peptide and Fluorescence 9
1.3 Noninvasive Imaging 12
1.4 Research Frame Work 14
Chapter 2 Materials and Methods 21
2.1 Materials 21
2.2 Equipment 22
2.3 Solution Formula 23
2.3.1 Phosphate Buffered Saline Solution (PBS), pH 7.4 23
2.3.2 DMEM-HG Culture Medium for WEHI 274.1 Monocytes 23
2.3.3 DMEM-HG Culture Medium for 3T3 Cells 23
2.3.4 DMEM-HG/F-12 Culture Medium for hASCs 24
2.3.5 MTT Assay Working Solution 24
2.3.6 Cyanine 5 Staining Solution 25
2.3.7 Oil Red O Staining Solution 25
2.3.8 Prussian Blue Staining Solution 25
2.4 Methods 26
2.4.1 Characterization 26
2.4.1.1 Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM) 26
2.4.1.2 Particle Size and Zeta Potential Analyzer 26
2.4.1.3 X-ray Photoelectron Spectroscope (XPS) and Magnetometer 26
2.4.2 in vitro Study 27
2.4.2.1 Cell Culture of WEHI 274.1 Monocytes 27
2.4.2.2 Cell Culture of 3T3 Cells and hASCs 27
2.4.2.3 MTT Assay 28
2.4.2.4 Live/Dead Assay 29
2.4.2.5 Nanoparticles Affinity Test 30
2.4.3 in vivo Study 31
2.4.3.1 Animal Model 31
2.4.3.2 Nuclear Magnetic Resonance Imaging (MRI) 32
2.4.3.3 Non-invasion in vivo Imaging System (IVIS) 32
2.4.3.4 Histology Staining 33
2.4.4 Statistical Analysis 33
Chapter 3 Results and Discussions 34
3.1 Characterization of Iron Oxide MNPs 34
3.1.1 Structural Property of Iron Oxide MNPs 34
3.1.2 Composition of Iron Oxide MNPs 36
3.1.3 Magnetic Measurements 38
3.2 Cytotoxicity and in vitro Study 43
3.2.1 Cytotoxicity and Cell Viability of MCP-1-motif MNPs 43
3.2.2 Specific Affinity of MCP-1-motif MNPs 44
3.3 in vivo Study, Imaging and Histology 50
3.3.1 Nuclear Magnetic Resonance Imaging (MRI) 50
3.3.2 Non-invasion in vivo Imaging System (IVIS) 52
3.3.3 Histology 54
Chapter 4 Conclusions 65
Chapter 5 Future Work 67
REFERENCES 68

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