臺灣博碩士論文加值系統

動脈粥狀硬化是全球導致心血管疾病以及死亡的主要原因之一，它是一種血管慢性發炎性疾病，主要導因於白血球所介導的血管內皮功能障礙，並促進富含脂肪酸的斑塊發展。在動脈粥狀硬化發展過程中，趨化因子能吸引單核球到血管壁上，促使單核球活化為成熟的巨噬泡沫細胞、誘導平滑肌細胞遷移和生長、以及進行血小板的活化和斑塊不穩定。
趨化因子之一的C-C motif chemokine ligand 4 (CCL4)也稱為巨噬細胞炎性蛋白1β（Macrophage inflammatory protein-1β；MIP-1β），目前被認為是自然殺手細胞，單核球以及其它免疫細胞的化學趨化物。研究顯示在動脈硬化斑塊中可見CCL4的表現；並在動脈粥狀硬化病人的血中也發現具有高濃度的CCL4，而具有較高濃度CCL4的病人，中風及心血管疾病的風險也比較高。因此本篇研究的目的就是去探討CCL4在動脈粥狀硬化所扮演的角色。
本篇研究假設CCL4之所以與中風有關是由於其會促使動脈粥狀硬化斑塊趨向不穩定的狀態發展，因此本篇研究使用apolipoprotein E knock out (ApoE KO)老鼠作為動物模組，評估當使用CCL4抗體抑制老鼠體內CCL4濃度時，對動脈粥狀硬化的影響。結果顯示在給予CCL4抗體的老鼠，血清中的發炎因子interleukin 6(IL-6)、tumor necrosis factor alpha (TNF-α)比起控制組有明顯下降，血脂也降低。其血管壁上的動脈粥狀硬化斑塊減少，所造成的斑塊具有較厚的纖維帽，及較少的巨噬細胞累積，並且其斑塊內的matrix metalloproteinase-2 (MMP-2)、matrix metalloproteinase-9 (MMP-9)較少，顯示其發展的動脈粥狀硬化斑塊較為穩定。
本篇研究的研究結果顯示CCL4可能對動脈粥樣化形成至關重要。CCL4可能是未來治療動脈粥樣硬化的潛在治療標靶。

Atherosclerosis, a chronic inflammatory disease, is a major cause of cardiovascular disease and death worldwide. It begins with endothelial dysfunction, followed by the activation of white blood cell, and leads to fatty-rich plaque formation. During development of atherosclerosis, chemokines attract monocytes into the arterial wall, promote maturation of monocytes to macrophage foam cells, induce growth and migration of smooth muscle cells, and conduct platelet activation, which may lead to plaque destabilization, resulting in plaque rupture and thrombosis formation as the major cause of ischemic stroke and acute myocardial infarction.
Chemokine (C-C motif) ligand 4 (CCL4), also known as macrophage inflammatory protein-1β (MIP-1β), has been regarded as a chemoattractant for natural killer cells, monocytes and a variety of other immune cells. Studies have indicated that CCL4 was expressed in atherosclerotic plaques. In addition, elevated levels of circulating CCL4 have been detected in atherosclerotic patients. Patients with the highest quartile of CCL4 showed a higher risk of stroke and cardiovascular events. Accordingly, we hypothesized that CCL4 might contribute to unstable plaque formation and facilitate the development of plaque rapture for clinical cardiovascular events.
The purpose of this study was to clarify the role of CCL4 in the development and progression of atherosclerosis. In the in vivo study, apolipoprotein E –knockout mice were used as an atherosclerosis model. Anti-CCL4 antibody was given to evaluate the in vivo effect of CCL4 inhibition on atheroma plaque formation and progression. In anti-CCL4 antibody treatment animals, the serum levels of IL-6 and TNF-α were reduced and the blood lipid profiles were modified in a dose-dependent fashion. Moreover, anti-CCL4 antibody inhibited plaque progression efficiently. Moreover, anti-CCL4 antibody could significantly reduce the size and prohibit the progression of atheroma plaques. Compared to that in control group, the plaques consist of a thicker fibrous cap, with reduced macrophage content and less MMP-2 and MMP-9 expression in anti-CCL4 antibody treatment animals, suggesting that anti-CCL4 antibody may suppress and stabilize the plaques.
The findings of this studies indicated that CCL4 may contribute to the development and progression of atheroma plaques. Given the critical role of unstable plaques in clinical cardiovascular events, CCL4 might be a potential therapeutic target for the prevention and treatment of atherosclerosis cardiovascular diseases.

Content …………………….………………………………...…………………..i
Acknowledgments…………………………………………………………………iii
Chinese abstract …………………………………………………………………..iv
English abstract …………………………………………………………………....v
Chapter 1. Introduction ………………………………………………………..1
1.1 Atherosclerosis ……………………………………………………………...1
1.2 Formation of Atherosclerotic Plaques ………………………………………2
1.3 Symptoms of Atherosclerosis ……………………………………………….2
1.4 Complications of Atherosclerosis …………………………………………...2
1.5 Risk Factors of Atherosclerosis……………………………………………...3
1.6 CC chemokine family and Atherosclerosis ………………………………....3
1.7 Chemokine (C-C motif) Ligand 4, CCL4 and Atherosclerosis ……………..4
1.8 C-C Chemokine Receptor Type 5, CCR5 and Atherosclerosis ……………..5
1.9 Aim ………………………………………………………………………….5
Chapter 2. Materials and Methods ……………………………………………6
2.1 Animal Model ………………………………………………………………6
2.2 Tissue Harvesting …………………………………………………………..6
2.3 Histologic Staining ………………………………………………………....6
2.4 Immunohistochemical Staining …………………………………………….7
2.5 Biochemical Indexes ……………………………………………………….7
2.6 Enzyme Linked Immunosorbant Assay ………………………………….....8
2.7 Western Blot ……………………………………………………………..…8
2.8 Statistical Analyses………………………………………………………….9
Chapter 3. Experimental Design ……………………………………………...10
Chapter 4. Results ……………………………………………………………..11
4.1 Elevated Levels of CCL4 in Serum and Aorta of Atherosclerotic Mice Was Reduced After 4 Weeks Antibody Treatment. ………………………...….11
4.2 CCL4 Neutralization Attenuated Pro-Inflammatory Factors in Circulation and Atherosclerotic Plaques. ……………………………………………...11
4.3 CCL4 Neutralization Effect on Metabolic Parameters. …………….….12
4.4 Effect of CCL4 Depletion on Atherosclerotic Plaque Development. ……….12
4.5 Effect of CCL4 Depletion on Atherosclerotic Plaque Quality. ……….……..12
Chapter 5. Discussion ………………………………………………………..…14
Chapter 6. Conclusion ……………………………………………………….....19
Chapter 7. References ………………………………………………………….20

Tables ……………………………………………….………………………….…..28
Table 1. Metabolic Data in Healthy and Atherosclerotic Mice ……….……28
Figures …………………………………………………………….……………….29
Figure 1. Anti-CCL4 Antibody Effect on Cytokine in Serum. ………………………………………………………………….29
Figure 2. Anti-CCL4 Antibody Effect on Metabolic Parameters. ………………………………………………………..…....30
Figure 3. Anti-CCL4 Antibody Reduces Atherosclerosis Lesion Size. ………………………………………………………………….....32
Figure 4. Anti-CCL4 Antibody Reduce Necrotic Area and Increase Fibrous Cap Thickness. ……………………..………………………………………33
Figure 5. Anti-CCL4 Antibody Reduce CCL4 Protein Expression in Aorta ……………………………………………………………………...34
Figure 6. Anti-CCL4 Antibody Reduce the Levels of Macrophages and CCL4 in Plaques. ……………………………….……………………………… 35
Figure 7. Anti-CCL4 Antibody Reduce MMP-2, MMP-9 Level in Plaques. ……………………………………………………………........37
Appendices…………………………………………………………….……………38

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