高效液相色谱法测定A549细胞中的胆固醇水平研究

摘要

目的 建立检测A549细胞内胆固醇水平的高效液相色谱(HPLC)方法,为以胞内胆固醇相对水平变化作为指标鉴定组织细胞泡沫化程度及特定组织细胞泡沫化对组织损伤提供技术支持.方法 用氢氧化钾乙醇溶液和三氯乙酸分别除去细胞裂解液中的三酰甘油和蛋白,用正己烷-异丙醇的混合溶剂提取细胞内的胆固醇.胆固醇水平采用HPLC法测定,色谱柱为ZORBAX SB-C18(4.6 mm×150.0 mm,5 μm),流动相为乙腈-异丙醇(67∶33,V/V),流速为1 mL/min,检测波长为206 nm.结果 细胞内胆固醇在5~100 ng/μL内线性关系良好(r=0.999 9);相对标准偏差(RSD)均低于3.0%,平均加样回收率为94.89%,RSD为2.86%(n=6).结论 HPLC法简便、准确、重复性好,可用于泡沫细胞程度鉴定,为探讨脂质代谢异常与器官损伤及进一步研究胆固醇代谢紊乱与肺损伤奠定基础.%Objective To establish a HPLC method for detecting intracellular cholesterol level in A549 cells so as to provide the technical support for the tissue injury by identifying the foaming degree of tissues cells with intracellular cholesterol relative level change as the indicator and specific tissue foaming.Methods Protein and triglycerides in the cell lysis solution were removed by using trichloroacetic acid and KOH ethanolic solution,the mixed solvent of normal hexane and isopropanol was used to extract intracellular cholesterol.The cholesterol level was detected by adopting the HPLC method.The chromatographic column was ZORBAX SB-C18(4.6 mm×150.0 mm,5 μm),the mobile phase was acetonitrile-isopropanol(67∶33,V/V),the flow rate was1 mL/min and the detection wavelength was at 206 nm.Results The linear range of intracellular cholesterol level showed good linearity in the range of 5-100 ng/μL(r=0.999 9).RSD was less than 3.0%.The average recovery rate was 94.89% and RSD was 2.86%(n=6).Conclusion The used method is convenient,simple and accurate with good repeatability,can be used to identify foam cell,and lays the foundation for exploring the lipid metabolism abnormality and organ injury and future studying the cholesterol metabolism disturbance and pulmonary injury.