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目的探讨体外氧化修饰后的高密度脂蛋白(ox-HDL)对人外周血单核细胞源性泡沫细胞内胆固醇流出的影响。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(LDL)及高密度脂蛋白(HDL),分别以Cu2+诱导法将其氧化修饰成ox-LDL及ox-HDL。采用密度梯度离心法和塑料吸附法从人外周血中分离出单核细胞,以50nmol/L佛波酯(PMA)刺激48h使之转化为巨噬细胞。细胞分为对照组(ox-LDL组)、阳性对照组(HDL组)及实验组(ox-HDL组),对照组仅加入终浓度为80mg/L的ox-LDL;HDL组和ox-HDL组分别先加入终浓度均为50mg/L的HDL和ox-HDL共孵育1h后,再分别加入终浓度为80mg/L的ox-LDL,并于实验的0、6、12及24h测定细胞内总胆固醇(TC)、游离胆固醇(FC)及蛋白(Pro)含量。观察不同时间点各组细胞内TC/Pro比值的变化,并比较ox-HDL与HDL对细胞内TC/Pro比值影响的时效关系。结果成功地由体外氧化修饰了LDL及HDL并制备出理想的泡沫细胞模型。在实验的6、12及24h,ox-HDL组细胞内TC/Pro比值均较HDL组细胞高,差异具有统计学意义(P<0.05)。结论体外氧化修饰后的HDL导致其胆固醇逆向转运(RCT)功能与效率的降低。
Objective To investigate the effect of oxidized high density lipoprotein (ox-HDL) on the efflux of cholesterol from human peripheral blood monocyte-derived foam cells. Methods LDL and HDL were isolated from plasma by one-step density gradient ultracentrifugation and oxidized to ox-LDL and ox-HDL by Cu2 + -induced method respectively. Mononuclear cells were isolated from human peripheral blood by density gradient centrifugation and plastic adsorption and stimulated with 50 nmol / L phorbol ester (PMA) for 48 h to transform them into macrophages. The cells were divided into control group (ox-LDL group), positive control group (HDL group) and experimental group (ox-HDL group), and the control group only received ox-LDL at the final concentration of 80 mg / Group were added to the final concentration of 50mg / L of HDL and ox-HDL co-incubated for 1h, then added to a final concentration of 80mg / L of ox-LDL, and in the experiment 0,6,12 and 24h determination of intracellular Total cholesterol (TC), free cholesterol (FC) and protein (Pro) content. The changes of intracellular TC / Pro ratio at different time points were observed. The effect of ox-HDL and HDL on the TC / Pro ratio was also observed. Results LDL and HDL were successfully modified by in vitro oxidation and the ideal foam cell model was prepared. The intracellular TC / Pro ratio of ox-HDL group was higher than that of HDL group at 6, 12 and 24 h of the experiment, the difference was statistically significant (P <0.05). Conclusion In vitro oxidative modification of HDL leads to a decrease in the function and efficiency of reverse cholesterol transport (RCT).