臺灣博碩士論文加值系統

　　停經後的婦女冠狀動脈心臟疾病的發生率較生殖期的婦女高。因此停經後婦女補充女性賀爾蒙被認為應能預防或降低冠狀動脈心臟疾病的發生率。許多臨床實驗及動物實驗有關賀爾蒙療法對心血管系統的保護作用並未有一致的結果。動脈粥狀硬化 (atherosclerosis)為引起冠狀動脈心臟疾病的主要原因。動脈粥狀硬化是一個慢性發炎反應疾病。在發展過程中，脂質堆積於血管的內膜(intima)中的巨噬細胞及平滑肌細胞。研究指出賀爾蒙替代療法能夠調控許多免疫發炎因子。巨噬細胞移動抑制因子 (MIF) 是一個具有多方面功能的因子，它能夠調控免疫及發炎反應；然而至今尚未見有關於賀爾蒙替代療法對於巨噬細胞移動抑制因子影響之研究。在本研究當中，我利用去卵巢的ApoE基因剔除小鼠去探討不同種類及不同劑量的荷爾蒙對於動脈粥狀硬化進展的影響，並進一步分析這些影響是否經由影響巨噬細胞移動抑制因子表現而達成的。實驗設計包括將去卵巢的ApoE基因剔除小鼠以口服的方式給予荷爾蒙，所使用的賀爾蒙包括結合型雌激素 (CEE)，雌激素 (E2)，醋酸甲羥孕酮 (MPA)。經16至20週後，犧牲動物，取其血清以測量血清中脂質及巨噬細胞移動抑制因子的含量。此外，取其主動脈藉由oil red-O染色的方式探討動脈粥狀硬化的程度，並利用免疫染色法偵測巨噬細胞移動抑制因子於動脈硬化斑中的表現量。實驗結果發現結合型雌激素或雌激素在給予相當於停經後婦女服用荷爾蒙的劑量之處理後能夠明顯地降低動脈粥狀硬化的面積，但對於血清中脂質的含量卻沒有影響。服用高劑量的雌激素不僅減少動脈粥狀硬化的面積，並且降低血清中膽固醇的含量。雌激素合併醋酸甲羥孕酮一起使用仍能夠降低動脈硬化的程度。此外，我們更進一步探討賀爾蒙對巨噬細胞移動抑制因子的表現。結果發現結合型雌激素、雌激素或是雌激素和醋酸甲羥孕酮能夠降低動脈硬化斑中巨噬細胞內巨噬細胞移動抑制因子表現量；血清中巨噬細胞移動抑制因子的含量在所有荷爾蒙處理之下卻沒有明顯性的改變。總而言之，我們的實驗結果呈現賀爾蒙可能會藉由降低巨噬細胞移動抑制因子於巨噬細胞中的表現量而達到對於心血管疾病的保護作用。

　　The incidence of coronary heart disease (CHD) is higher in postmenopausal women than that in women at reproductive age. Hormone therapy (HT) has been used to relieve menopausal syndrome and is expected to have protective effect on cardiovascular disease. However, clinical trials have provided controversial results on the effect of HT on cardiovascular disease. Atherosclerosis is a chronic inflammatory disease characterized by accumulation of lipid in macrophages and smooth muscle cells in the intima of blood vessels. Studies have shown that HT modulates inflammatory mediators. Macrophage migration inhibitory factor (MIF), a pleiotropic cytokine that regulates inflammatory and immune responses, is also detected in atherosclerotic lesions. It has been reported that MIF is expressed in all stage of atherosclerosis. However, no information related to HT on the role of MIF in atherosclerosis has been reported yet. In this study, we used apolipoprotein E deficient (ApoE-/-) mice to investigate whether estrogen and progesterone might affect atherosclerotic progression and whether these effects were mediated by MIF. Ovariectomized mice were fed high cholesterol diet and received oral administration of conjugated equine estrogen (CEE), 17β-estradiol (E2), medroxyprogesterone acetate (MPA) or E2 combined with MPA. After 16~20 weeks, mice were sacrificed, and their sera were collected for the measurement of lipid and MIF levels. The aortae were removed for the examination of lipid deposition by using oil red O staining. MIF expression in macrophages and smooth muscle cells of atherosclerotic plague were detected by double immunostaining. Our results showed that CEE or E2 at the dose used by postmenopausal women significantly reduced atherosclerotic lesion area without affecting plasma lipid levels. The combination of E2 with MPA also reduced atherosclerotic lesion area. The treatment of E2 at high dose reduced both plasma cholesterol level and atherosclerotic lesion area. It indicates that factors other than lowering serum lipid levels may also be involved in hormonal regulation of atherosclerotic progression. Our immunostaining results showed that MIF was expressed in macrophages and smooth muscle cells of atherosclerotic plaques. The treatment of CEE, E2 and E2 combined with MPA significantly reduced MIF expression in macrophage but not in smooth muscle cells. In contrast, serum levels of MIF did not change under all various treatments of hormones. All together, our results suggest that the reduction of MIF in macrophages may also contribute to the protective effects of hormones on cardiovascular disease.

中文摘要 1
ABSTRACT 3
誌謝 5
TABLE OF CONTENTS 7
LIST OF FIGURE 9
LIST OF TABLES 11
ABBREVIATIONS 12

INTRODUCTION 13
OVARIAN HORMONES 13
MENOPAUSE AND HORMONE THERAPY 13
EFFECTS OF HORMONE THERAPY ON THE RISK OF CARDIOVASCULAR DISEASE 15
PATHOGENESIS OF ATHEROSCLEROSIS 17
MACROPHAGE MIGRATION INHIBITORY FACTOR (MIF) 18
MIF and atherosclerosis 19
ATHEROSCLEROSIS IN APO E DEFICIENT MICE 20
Murine cholesterol metabolism and apolipoprotein E 20
Plaque pathology in the ApoE-/- mice 20
OBJECTIVES OF THE STUDY 22

MATERIALS AND METHODS 23
ANIMALS 23
STUDY DESIGN 23
ISOLATION OF GENOMIC DNA 24
POLYMERASE CHAIN REACTION (PCR) 25
QUANTITATION OF ATHEROSCLEROSIS 25
DOUBLE IMMUNOFLUORESCENCE STAINING 26
SERUM LEVELS OF MIF 27
LIPOPROTEIN MEASUREMENT 27
STATISTICAL ANALYSIS 28

RESULTS 29
PART I: EFFECTS OF HORMONES ON ATHEROSCLEROTIC PROGRESSION 29
CEE decreased atherosclerotic progression 29
E2 and E2 plus MPA treatment decreased atherosclerotic progression in ApoE-/- mice 30
PART II: EFFECTS OF HORMONES ON LIPID LEVELS IN SERUM 31
CEE did not decrease serum levels of lipids 31
E2 at the dose used by postmenopausal women did not change serum levels of lipids 31
High dose of E2 and E2 plus MPA treatment affected serum levels of lipids 32
PART III: EFFECTS OF HORMONES ON EXPRESSION OF MIF 32
CEE decreased the expression of MIF in atherosclerotic plaques 32
E2 and E2 plus MPA decreased MIF expression in atherosclerotic plagues 33
Hormones did not significantly affect serum levels of MIF 34

DISCUSSION 35
REFERENCES 59
APPENDIX 62
ABOUT THE AUTHOR 68

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