HPLC法测定apoA-1介导的泡沫细胞胆固醇流出的实验研究  

Research on the determination of cholesterol efflux from foam cells mediated by apoA-1 using HPLC analysis

作　　者：马卫列[1] 龚晓华[1] 李观强[2] 丁航[1] 兰柳波[1] 陈小谊[1] 张志珍[1] 

机构地区：[1]广东医学院生物化学与分子生物学教研室,广东东莞523808 [2]深圳市龙岗区人民医院检验科,广东深圳518172

出　　处：《重庆医学》2015年第29期4116-4119,4122,共5页Chongqing medicine

基　　金：国家自然科学基金资助项目(81170267);广东省自然科学基金资助项目(S2011010002984);东莞市高等院校科研机构科技计划项目(2012108102037)

摘　　要：目的建立用高效液相色谱(HPLC)法测定载脂蛋白A-1(apoA-1)介导的泡沫细胞内胆固醇流出率的实验方法。方法人单核细胞THP-1用160nmol/L佛波酯(PMA)诱导24h分化为贴壁巨噬细胞(PMA组),再用50μg/mL乙酰化低密度脂蛋白(ac-LDL)处理48h,诱导巨噬细胞变成泡沫细胞(ac-LDL组);加入含apoA-1的1640培养基培养24h(apoA-1组)。油红O染色和HPLC法测定细胞内胆固醇水平,鉴定巨噬细胞源性泡沫细胞模型。采用HPLC分析和液体闪烁计数法测定apoA-1介导的泡沫细胞内胆固醇流出率。结果 THP-1巨噬细胞源性泡沫细胞经油红O染色后,细胞内可明显观察到大量红色脂滴存在。胆固醇水平测定显示,ac-LDL组内游离胆固醇、总胆固醇和胆固醇酯的水平明显高于PMA组(P<0.01)。ac-LDL处理细胞48h后,ac-LDL组内总胆固醇水平为80.25μg/mg细胞蛋白,胆固醇酯水平为47.65μg/mg细胞蛋白,占总胆固醇的59.38%,与PMA组相比,差异有统计学意义(P<0.01)。HPLC分析和液体闪烁计数法结果表明,apoA-1介导泡沫细胞内apoA-1组胆固醇流出率分别为5.63%和7.08%(P<0.01)。结论本研究成功建立了酶促反应结合HPLC测定泡沫细胞内胆固醇流出的方法,为后续研究细胞脂质代谢提供了实验基础。Objective To establish a high performance liquid chromatography（HPLC）method for determining the cholesterol efflux from macrophage-derived foam cells mediated by apolipoprotein A-1（apoA-1）.Methods Human THP-1 monocytic cells,pre-treated with 160nmol/L phorbol-12-myristate acetate（PMA）for 24 hto differentiate into adherence macrophages,then incubated with 50μg/mL acetylated low density lipoprotein（ac-LDL）for 48 hto induce foam cells formation,then added apoA-1for 24 h.THP-1-derived macrophage foam cells were identified by oil red O-staining,and the cellular cholesterol content by measured by HPLC method.Cholesterol efflux from macrophage foam cells was determined by HPLC analysis and liquid scintillation counting,respectively.Results Oil red O-stainable lipid droplet accumulation were observed in entire cytoplasm of THP-1-derived macrophage foam cells.Measuring cellular cholesterol content showed that free cholesterol,total cholesterol and cholesterol ester content in macrophage foam cells were increased remarkably than PMA group macrophages（P〈0.01）.After treated with ac-LDL for 48 h,the macrophage foam cells accumulated 80.25μg/mg cell protein and 47.65μg/mg cell protein respectively,and the cholesterol ester accounted for 59.38% of the cellular total cholesterol（P〈0.01）.The ratio of cholesterol efflux reached 5.63% and7.08% respectively by HPLC analysis and liquid scintillation counting using apoA-1mediation（P〈0.01）.Conclusion Combination of an enzymatic catalysis and HPLC method for determining cholesterol efflux from foam cells is successfully established in this study,thus providing a technical foundation for the further study of cellular lipid homeostasis.

关 键 词：色谱法 高效液相 泡沫细胞 载脂蛋白A-1 胆甾烯酮 胆固醇流出 

分 类 号：R543.5[医药卫生—心血管疾病]