Pitas R E, Innerarity T L, Weinstein J N, Mahley R W
Arteriosclerosis. 1981 May-Jun;1(3):177-85. doi: 10.1161/01.atv.1.3.177.
We have developed a procedure for labeling lipoproteins with the fluorescent probe 3,3'-dioctadecylindocarbocyanine (Dil) and have used Dil-labeled native and acetoacetylated lipoproteins to differentiate macrophages from fibroblasts in mixed cell culture. Lipoproteins labeled with this probe were suitable for the direct viewing of their binding and internalization by cells in vitro. The labeling technique has been applied to human low density lipoproteins (LDL) and to two canine cholesterol-induced lipoproteins: apo-E HDLc, which contain only the E apoprotein (apo-E), and beta-migrating, very low density lipoproteins (beta-VLDL), which contain apo-B and apo-E. The Dil-labeled lipoproteins showed specific high affinity binding to human fibroblasts via the LDL (apo-B, -E) receptors. The equilibrium dissociation constant for the binding of Dil-labeled apo-E HDLc and LDL were the same as for the respective native lipoproteins. The specific binding of Dil-labeled LDL and apo-E HDLc was further substantiated by fluorescence microscopy. When an excess of native (non-fluorescent) lipoproteins was added to the Dil-labeled lipoproteins, essentially no fluorescently labeled lipoproteins were seen associated with the cells. The Dil-labeled LDL, apo-E HDLc, and beta-VLDL, which were bound to the cells at 4 degrees C, were associated with the cell surface and were often observed in linear arrays. Cells that were either incubated with Dil-labeled lipoproteins at 4 degrees C and subsequently heated to 37 degrees C or incubated with the Dil-labeled lipoproteins at 37 degrees C showed internalized perinuclear fluorescence. When Dil-labeled LDL, apo-E HDLc, or beta-VLDL were treated with diketene to acetoacetylate their lysine residues, and then were incubated at 37 degrees C with mixtures of fibroblasts and mouse peritoneal macrophages in culture, the fibroblasts did not become fluorescently labeled. The macrophages became highly fluorescent, however. The acetoacetylation inhibited the interaction of the lipoproteins with the apo-B, -E receptors of fibroblasts and stimulated their uptake by macrophages. The use of fluorescently labeled native lipoproteins and chemically modified lipoproteins may allow the functional differentiation of macrophages from other cell types (e.g., fibroblasts and smooth muscle cells) in the arterial wall. This differentiation may be useful in determining the origin of the lipid-laden foam cells of atherosclerotic lesions.
我们已经开发出一种用荧光探针3,3'-二辛基吲哚羰花青（Dil）标记脂蛋白的方法，并使用Dil标记的天然脂蛋白和乙酰乙酰化脂蛋白在混合细胞培养物中区分巨噬细胞和成纤维细胞。用该探针标记的脂蛋白适用于在体外直接观察其与细胞的结合及内化过程。该标记技术已应用于人类低密度脂蛋白（LDL）以及两种犬类胆固醇诱导脂蛋白：仅含E载脂蛋白（apo-E）的apo-E HDLc和含apo-B和apo-E的β迁移极低密度脂蛋白（β-VLDL）。Dil标记的脂蛋白通过LDL（apo-B、-E）受体与人成纤维细胞表现出特异性高亲和力结合。Dil标记的apo-E HDLc和LDL结合的平衡解离常数与各自的天然脂蛋白相同。荧光显微镜进一步证实了Dil标记的LDL和apo-E HDLc的特异性结合。当向Dil标记的脂蛋白中加入过量的天然（非荧光）脂蛋白时，基本上看不到有荧光标记的脂蛋白与细胞相关联。在4℃下与细胞结合的Dil标记的LDL、apo-E HDLc和β-VLDL与细胞表面相关联，且常呈线性排列。在4℃下用Dil标记的脂蛋白孵育随后加热至37℃的细胞，或在37℃下用Dil标记的脂蛋白孵育的细胞均显示核周荧光内化。当用双乙烯酮处理Dil标记的LDL、apo-E HDLc或β-VLDL使其赖氨酸残基乙酰乙酰化，然后在37℃下与培养的成纤维细胞和小鼠腹腔巨噬细胞混合物孵育时，成纤维细胞未被荧光标记。然而，巨噬细胞却变得高度荧光化。乙酰乙酰化抑制了脂蛋白与成纤维细胞的apo-B、-E受体的相互作用，并刺激巨噬细胞对其摄取。使用荧光标记的天然脂蛋白和化学修饰的脂蛋白可能有助于在动脉壁中从其他细胞类型（如成纤维细胞和平滑肌细胞）中功能性区分巨噬细胞。这种区分对于确定动脉粥样硬化病变中富含脂质的泡沫细胞的起源可能是有用的。
