体外氧化修饰高密度脂蛋白对人单核细胞源性泡沫细胞内胆固醇流出的影响

Effects of in vitro oxidized HDL on cholesterol efflux of human monocyte-derived foam cells

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。

AIM To study the effects of oxidized high density lipoprotein （ox-HDL） on cholesterol efflux of human monocyte-derived foam cells. METHODS Low density lipoprotein （LDL） and high density lipoprotein （HDL） were isolated from human plasma by one time density gradient centrifugation, and Cu^2＋ at concentration of 10 μmol/L was used to induce the oxidation of LDL （ox-LDL）. Monocytes were isolated from human peripheral blood by Ficoll-Hypaque density gradient centrifugation and plastic adsorptive process. The isolated cells were induced and stimulated by phorbol 12-myristate 13-acetate （PMA） at concentration of 50 nmol/L for 48 h to transfer them to macrophages. The cells were then allocated to control group （ ox-LDL group ）, positive control group （ HDL group ） and experimental group （ox-HDL group）, ox-LDL alone at a final concentration of 80mg/L was added to control group, while HDL and ox-HDL at 50 mg/L were co-incubated in HDL group and ox-HDL group for 1h, followed by addition of ox-LDL at a final concentration of 80mg/L to both groups. The total cholesterol （ TC ） , free cholesterol （FC） and protein （Pro） in cultured cells were detected at 6, 12 and 24 h of culture. The ratio of TC to Pro in cells at different time points was observed and the time-effect relationship of HDL and ox-HDL to cells was compared. RESULTS The isolated LDL and HDL were successfully oxidative modified in vitro and a human peripheral blood monocyte-derived foam cell model was established. Ratio of TC to Pro at 6, 12 and 24 h in ox-HDL group was significantly higher than that of HDL group （P 〈 0.05）. CONCLUSION The reverse cholesterol transport （RCT） capacity and efficiency of HDL in cells decrease greatly following oxidization in vitro.