臺灣博碩士論文加值系統

動脈粥狀硬化疾病已成為許多開發國家中，主要罹病及死亡率的主因。動脈粥狀硬化疾病的主要致病機轉是血管壁上所引起的發炎反應。目前用於治療動脈粥狀硬化的藥物開發策略包括了老藥新用及研發全新的的藥劑。
降血脂藥物施德丁是目前最普遍使用於治療動脈粥狀硬化的藥物。現今的研究已論證施德丁(statin)除了降血脂外，亦具有其他的功能。然而目前少有研究顯示施德丁對於動脈粥狀硬化基因表現的影響，故我們利用餵食高膽固醇飼料的自發性動脈粥狀硬化缺陷小鼠模式(apoE-deficient mice)並給予施德丁的治療;以利研究施德丁對於動脈粥狀硬化基因表現的影響。我們以安捷倫(Agilent)公司的晶片進行寡核苷酸微陣列(oligonucleotide microarray) 研究美百樂鎮錠(pravastatin)對主動脈基因表現的影響。我們分析20281個小鼠基因，發現在美百樂鎮錠與對照組間，共有94個基因表現具顯著差異。其中有30個基因為高表現者;有60個基因為低表現者。我們期望此研究結果能提供研究施德丁相關分子機制的方向。
巨噬細胞在動脈粥狀硬化發炎反應中扮演重要的角色。鴉片類胜肽(opioid peptides)已被報導具有調節免疫反應的能力。納洛克松(naloxone)和右甲嗎喃(dextromethorphan)都是嗎啡(morphinan)的類似物;它們兩者都被發現可抑制神經系統中的巨噬細胞-微神經膠細胞(microglia)的活性。故探討納洛克松和右甲嗎喃可否經由降低巨噬細胞的發炎反應，進而減緩動脈粥狀硬化的形成。在我們的研究結果中發現納洛克松和右甲嗎喃;不論在細胞或動物體內都可顯著的降低發炎因子及過氧化物(superoxide)的生成。我們亦發現納洛克松和右甲嗎喃確實可減緩自發性動脈粥狀硬化缺陷小鼠及頸動脈結紮手術中動脈粥狀硬化的情況。納洛克松和右甲嗎喃是否是藉由抑制還原態的烟鹼醯胺腺嘌吟二核苷酸磷酸氧化酶(NADPH oxidase)，而減緩動脈粥狀硬化的情形則須要更深入的研究。

Atherosclerotic vascular disease is the major cause of morbidity and mortality in many developed countries. Central to the pathogenesis of atherosclerosis is the inflammation in the arterial wall. New approaches to atherosclerosis-related diseases include novel uses of proven treatments and development of innovative agents.
Statins are the most commonly prescribed agents for the treatment of hypercholesterolemia because of their efficacy in reducing low-density lipoprotein (LDL). Recent experimental and clinical evidence indicates the cholesterol-independent effects of statins. However, the genetic expression pattern changes in atherosclerotic lesions produced by statins are rarely studied. Cholesterol-fed apolipoprotein (apo) E-deficient mice were examined for the treatment effect of statin on aortic gene expression. The aortic gene expression affected by pravastatin was identified by the oligonucleotide microarray technology with Agilent gene chips. Microarray analysis of the expression of 20,281 murine genes in the aortas between the two groups indicated that 94 genes were significantly regulated. Thirty genes were up-regulated and 64 genes were down-regulated. Our study might provide insight into the clinical benefits of chronic statin treatment.
Macrophages play an important role in the inflammatory process in atherosclerosis. Opioid peptides have been shown to modulate immunoresponse. Naloxone is a non-selective antagonist of the opioid receptors. Dextromethorphan (DM) is the d-isomer of the codeine analog levophanol, a dextrorotatory morphinan. It is widely used as a cough suppressant in cold and cough medications with a high safety profile. Both of them could inhibit activation of microglia, the resident macrophage in nervous systems. We investigated whether naloxone and DM could reduce macrophage activation and influence atherosclerotic lesion formation in mice. In our study, we found that naloxone and DM pretreatment significantly suppressed the production of proinflammatory factors and superoxide in macrophage and mice after stimulation. The novel anti-inflammatory effect of naloxone and DM reduced the spontaneous aortic atherosclerotic lesion formation in apoE-deficient mice and the carotid neointima formation in C57BL6 mice receiving carotid ligation. The study of naloxone and DM generate many new and exciting hypotheses to be tested in the future.

Abstract -------------------------------------------------------------------- 1
Abstract in Chinese---------------------------------------------------------- 3
Introduction
Ⅰ. Atherosclerosis-an inflammatory disease---------------------------------- 4
Ⅱ. The step of atherosclerosis---------------------------------------------- 4
Endothelial dysfunction: setting the stage for inflammation ------------ 4
Fatty streak and vulnerable plaque formation---------------------------- 6
Ⅲ. Oxidative stress in atherosclerosis --------------------------------------6
The oxidative modification hypothesis of atherosclerosis-----------------6
Reactive oxygen species (ROS) and the phagocytic NAD(P)H oxidase---------8
Ⅵ. Animal models of experimental atherosclerosis-----------------------------9
Gene targeting atherosclerotic mouse models-----------------------------10
Carotid Intima-media thickening mouse models----------------------------12
Ⅴ. New trends in anti-atherosclerotic drugs---------------------------------14
Cholesterol-lowering drugs----------------------------------------------14
Novel anti-inflammatory drugs-------------------------------------------17
Objection of the study-------------------------------------------------------19
Materials and Methods
I. Animal and animal diet----------------------------------------------------20
II. Preparation of drugs-----------------------------------------------------20
III. Methods-----------------------------------------------------------------21
1. Human acute monocytic leukemia cell culture-------------------------------21
2. Evaluation of cell viability----------------------------------------------21
3. Isolation of LDL and Cu2+-Induced oxidation of LDL------------------------22
4. Evaluation of pro-inflammatory factors in THP-1 cells---------------------22
5. Evaluation of superoxide in THP-1 cells-----------------------------------22
6. Evaluation of NADPH oxidase activity in THP-1 cells-----------------------23
7. Determination of serum lipid profiles-------------------------------------23
8. Evaluation of lipid deposition in aorta-----------------------------------24
9. Isolation of the aorta RNA------------------------------------------------24
10.Oligonucleotide expression arrays----------------------------------------25
11. Real-time quantitative PCR (RT-PCR) -------------------------------------25
12. Determination of TNF-α production in mice--------------------------------26
13. Determination of PMN infiltration index in the lung----------------------26
14. Determination of superoxide production in the PMN------------------------26
15. Determination of superoxide production in the aorta----------------------27
16. Measurement of aortic superoxide production with DHE---------------------27
17. Mouse vascular remodeling model -----------------------------------------28
18. Statistical analyses-----------------------------------------------------28

Results and Discussion
Chapter 1. The effect of statin on the aortic gene expression profiling
I. Results-------------------------------------------------------------------29
1.Serum cholesterol and aortic atherosclerotic lesion------------------------29
2.Oligonucleotide expression arrays and real-time quantitative RT-PCR -------29
II. Discussion---------------------------------------------------------------31

Chapter 2. A novel inhibitory effect of naloxone on macrophage activation and atherosclerosis formation in mice
I. Results-------------------------------------------------------------------35
1. Naloxone treatment reduces macrophage cytokine production-----------------35
2. Naloxone treatment reduces TNF-α and superoxide production in mice--------36
3. Naloxone treatment inhibits atherosclerosis and neointima Formation-------36
II. Discussion---------------------------------------------------------------38

Chapter 3. Dextromethorphan is a novel inhibitor of inflammation that reduces
atherosclerosis and neointima formation in mice
I. Results-------------------------------------------------------------------41
1. Dextromethorphan treatment reduces macrophage cytokine production---------41
2.Dextromethorphan treatment reduces macrophage superoxide production--------42
3.Dextromethorphan treatment reduces macrophage NADPH oxidase activity-------42
4.Dextromethorphan treatment reduces superoxide production in mice-----------43
5.Dextromethorphan treatment inhibits atherosclerosis and neointima formation-------------------------------------------------------------------------------43
II. Discussion---------------------------------------------------------------45
Conclusion of the study------------------------------------------------------48
References-------------------------------------------------------------------49
Tables-----------------------------------------------------------------------67
Figures----------------------------------------------------------------------70
Publication list-------------------------------------------------------------88

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