亲环素A在巨噬细胞荷脂过程中的表达及普罗布考干预的影响

目的观察亲环素A在氧化型低密度脂蛋白诱导的巨噬细胞荷脂过程中的表达变化及其与细胞内胆固醇蓄积的关系,进而探讨亲环素A在动脉粥样硬化形成中的作用机制。方法采用75 mg/L氧化型低密度脂蛋白与RAW264.7巨噬细胞共同孵育,建立巨噬细胞荷脂化模型,油红O染色观察细胞内脂滴的形成,高效液相色谱法检测细胞内胆固醇含量,Western-blot检测亲环素A的蛋白表达;用75 mg/L普罗布考干预对上述检测的影响。结果氧化型低密度脂蛋白与RAW264.7细胞共同孵育48 h后,油红O染色显示细胞内有大量脂滴形成,细胞内总胆固醇和游离胆固醇含量均明显增加,胆固醇酯/总胆固醇的比值为42.3%±5.9%,符合荷脂细胞特征;亲环素A的蛋白表达明显减弱。予普罗布考处理,细胞内脂滴明显减少,胆固醇酯/总胆固醇的比值降至32.9%±2.5%,亲环素A的蛋白表达增加81.3%±3.6%,较对照组差异有显著性(P均<0.05)。结论氧化型低密度脂蛋白诱导的RAW264.7巨噬细胞荷脂过程中,亲环素A表达下调,促进细胞胆固醇蓄积;普罗布考干预可上调亲环素A蛋白表达,减轻细胞内的胆固醇蓄积。

Expression of Cyclophilin A in Lipid-Loaded Macrophages Induced by Oxidized Low Density Lipoprotein and the Effect of Probucol on Cellular Cholesterol Accumulation

Aim To observe the protein expression of cyclophilin A in lipid-loaded macrophages induced by oxidized low density lipoprotein(ox-LDL) and its effect on cellular cholesterol accumulation. Methods RAW264.7 mouse macrophages were treated with 75 mg/L ox-LDL for 0,24,48 and 72 hours.Oil red O dye experiment was used to show the lipid droplets in lipid-loaded macrophages. High performance liquid chromatography(HPLC) analysis was peformed to determine the content of cellular total cholesterol(TC) and cholesterol ester(CE).Westernblot analysis was used to detect the protein expression of cyclophilin A.Then,75 mg/L Probucol was added in the cells treated with ox-LDL for 48 hours. Results With ox-LDL treatment after 48 hours,Oil red O dye showed a lot of lipid droplets accumulated in RAW264.7 macrophages,HPLC analysis showed the content of cellular TC and CE were increased greatly and Western-blot analysis showed the expression of cyclophilin A was significantly decreased.However,with Probucol treatment,cyclophilin A was greatly increased,the lipid droplets significantly decreased,and the content of TC and CE also decreased. Conclusion The protein expression of cyclophilin A was down-regulated in lipid-loaded macrophages while the content of cellular TC and CE were increased greatly.However,Probucol could up-regulate the cyclophilin A expression and decrease the cellular cholesterol accumulation.