人外周血单核细胞源性泡沫细胞模型的建立

Establishment of a human peripheral blood monocyte-derived foam cell model

目的建立人外周血单核细胞源性泡沫细胞模型,为研究动脉粥样硬化机制及其防治提供新途径。方法采用一次性密度梯度超速离心法从血浆中分离低密度脂蛋白,以Cu2+诱导法将其氧化修饰成氧化低密度脂蛋白。采用密度梯度离心法和塑料吸附法从冠心病患者外周血中分离出单核细胞,首先以50 nmol/L佛波酯刺激48 h使之转化为巨噬细胞,然后与80 mg/L氧化低密度脂蛋白共孵育24 h,使之转化为泡沫细胞。结果经50 nmol/L的佛波酯诱导48 h后,光镜下发现单核细胞已转化为典型的巨噬细胞。油红O染色发现,与80 mg/L氧化低密度脂蛋白共孵育24 h后培养细胞中出现了大量的红染颗粒。高效液相色谱法测定细胞内胆固醇提示细胞内总胆固醇明显增加,其中胆固醇酯含量大于游离胆固醇,符合泡沫细胞的定义。结论直接采用冠心病患者外周血单核细胞成功地建立了泡沫细胞疾病模型,其病理生理学特性接近于体内状况,可为研究动脉粥样硬化机制和药物防治提供一种可靠的病理细胞模型。

Objectives To establish a human peripheral blood monocyte-derived foam cell model, so as to offer a new method for the study of the pathogenesis and advance mechanisms as well as the prevention and cure ways of atherosclerosis（AS）. Methods Low density lipoprotein （LDL） was isolated from human plasma by one time density gradient centrifugation, and Cu^2＋ at a concentration of 10 μmol/L was used to induce the oxidation of LDL （ox-LDL）. Mononuclear cells were isolated from human peripheral blood by Ficoll-Hypaque density gradient centrifugation and plastic adsorptive process. The isolated ceils were induced and stimulated by phorbol 12- myristate 13-acetate （PMA） in a concentration of 50 nmol/L for 48 h so as to make them transfer to macrophages. The successfully transferred cells were then co-incubated with ox-LDL at a concentration of 80 mg/L. Inverted microscope and oil red O staining technique were then used to identify the formation of foam ceils. Results After inducing by PMA at 50 nmol/L for 48 h, the isolated monocytes were successfully transferred to macrophages. When co-incubating with ox-LDL at 80 mg/L for 24 h, a great deal of lipid droplets could be observed in the cultured ceils through oil red O staining technique. The results by HPLC analysis also showed that when coincubated with ox-LDL at 80 mg/L for 24 h, the cholesterol in cells increased significantly, and the ratio of CE to TC （CE/TC） was beyond 60%, indicating the formation of foam cells. Conclusions A foam cell model was successfully established by the use of peripheral blood monocyte from cases with coronary heart disease. The pathophysiological characteristics of the established model were some more similar to that of cases with coronary heart disease than other cell line derived models, thus providing an even more real and reliable cell model for the study of the pathogenesis mechanisms and the prevention and cure ways of atherosclerosis.