應用「螢光顯微成像」及「拉曼光譜」技術探討高膽固醇血脂斑馬魚之血管脂肪斑塊

Abstract

冠狀動脈粥狀硬化肇因於血管壁沉積脂肪斑塊，嚴重時血管內徑會變窄，造成血液供給不足及心肌缺氧。若血管壁的脂肪斑塊發生破裂，血液會凝集成血塊，阻塞冠狀動脈，導致心肌梗塞。餵食兔子或老鼠高膽固醇食物為常見的動脈粥狀硬化動物模型。由於光學穿透度的限制，過去研究很少能在活體動物以非侵入的方式直接觀察脂肪斑塊。以斑馬魚研究動脈粥狀硬化的優點為其在幼魚階段身體接近透明，有機會能在活體以光學成像及光譜技術分析血管壁的脂肪斑塊。我們根據文獻的方法建立斑馬魚初期動脈粥狀硬化模型。藉由餵食螢光膽固醇酯，我們應用雷射共焦螢光顯微鏡對斑馬魚血管內之脂肪斑塊成像並透過影像分析加以定量。我們也成功應用拉曼顯微光譜技術量測血管內單顆脂肪斑塊的光譜。藉由餵食同位素膽固醇，我們證明血管壁的脂肪斑塊是來自於餵食之膽固醇。我們也發現斑馬魚血管壁之脂肪斑塊與氧化型低密度脂蛋白具有相似之拉曼光譜特徵。隨著餵食高膽固醇飼料時間增加，除了1155 cm-1及 1520 cm-1 的拉曼光譜線消失之外，1265 cm-1 及 1660 cm-1 的拉曼光譜線之強度也逐漸下降，此與低密度脂蛋白氧化程度上升之拉曼光譜變化趨勢一致。我們也探討降膽固醇藥物對斑馬魚血管壁脂肪斑塊之影響。實驗結果顯示，餵食添加普羅布考 (probucol) 的飼料能有效降低脂肪斑塊之累積，也能有效抑制形成氧化型脂肪斑塊，但卻無法將氧化型脂肪斑塊轉變為未氧化的脂肪斑塊。此研究首次展現應用拉曼散射光譜技術，探討降血脂藥物對斑馬魚血管壁脂肪斑塊化學結構之影響。藉由檢測斑馬魚脂肪斑塊的堆積及氧化程度，未來可以應用於快速評估新開發藥物治療動脈粥狀硬化之成效。

Lipid deposits formed on the surface of coronary arteries, termed atherosclerotic plaques, can cause narrowing of lumen or completely occlude the blood stream, and may eventually lead to heart hypoxia and coronary artery heart diseases. Rupture of vulnerable atherosclerotic plaques accounts for the majority of acute cardiovascular events. Excessive hyperlipidemia induced in mice and rabbits has been extensively employed to model human atherosclerosis, but microscopic exploration of the plaques is achieved mainly postmortem. Hypercholesterolemic zebrafish induced by high-cholesterol diet (HCD) has recently been reported to be a suitable disease model to imitate the early pathological process of atherosclerosis. Herein we report the first attempt on in vivo Raman microspectroscopic investigation of vascular lipids deposited in hypercholesterolemic zebrafish. Guided with fluorescence imaging, we have successfully obtained Raman spectra of individual vascular fatty plaques, and found that the 1155 cm-1 and 1520 cm-1 Raman bands attenuated completely whereas the 1265 cm-1 and 1660 cm-1 bands were moderately declined; these two characteristic features are profoundly consistent with that measured on oxidized low-density lipoproteins (LDL). To explore pharmacological applications of our approach, we examined also HCD-fed zebrafish that were treated with a cholesterol-lowering drug. The treatment of probucol to HCD-fed zebrafish reduced the total amount of vascular lipids, and, more significantly, the percentage of oxidized lipids; 60 % of the total fatty plaques examined in this work became non-oxidized in sharply contrast with the all oxidized fatty plaques found in the control without the treatment of probucol. Further mechanistic studies show that the treatment of probucol suppressed only the formation of oxidized fatty plaque but not reversed the oxidized lipid into non-oxidized one. We anticipate our approach is extendible to investigate the efficacy of therapeutic and diet interventions to atherosclerosis.