血清-巨噬细胞间胆固醇净流动测定研究  

Measurement of cholesterol net flux between serum and macrophage cells

作　　者：王默[1] 国汉邦[1] 赵海建[2] 杨睿悦[1] 李红霞[1] 王抒[1] 董军[1] 陈文祥[2] 

机构地区：[1]卫生部北京医院卫生部北京老年医学研究所卫生部老年医学重点实验室,100730 [2]卫生部临床检验中心

出　　处：《中华检验医学杂志》2013年第2期151-155,共5页Chinese Journal of Laboratory Medicine

基　　金：国家自然科学基金资助项目（81171647）

摘　　要：目的建立一种高效液相色谱（HPLC）测定血清-巨噬细胞间胆固醇净流动的方法，用以分析与动脉粥样硬化危险因素的关系。方法培养小鼠巨噬细胞J774，用含5％个体血清的培养基进行孵育8h，HPLC测定孵育前后细胞胆固醇，计算个体血清介导的细胞胆固醇净流动，建立血清-巨噬细胞间胆固醇净流动的测定方法。2010年10至12月征集75名北京医院健康志愿者，男38例、女37例，采集空腹静脉血，用所建方法测定胆固醇净流动；Spearman相关分析胆固醇净流动与血脂、脂蛋白、超敏C反应蛋白（hsCRP）等动脉粥样硬化危险因素的关系。结果发现个体血清孵育细胞时导致细胞胆固醇内流，细胞胆固醇随培养基中血清浓度和孵育时间的增加而增多。HPLC测定细胞胆固醇净流动的平均批内变异和总变异分别为0．81％-4．03％和4．25％-6．58％。75例健康志愿者胆固醇净流动均值为10．27μg／mg蛋白，标准差2．69μg/mg蛋白，中位数10．12μg/mg蛋白。胆固醇净流动与血清总胆固醇（TC）、三酰甘油（TG）、hsCRP，载脂蛋白（Apo）CⅡ、ApoCⅢ、小而密低密度脂蛋白胆固醇（LDLb-C）、高密度脂蛋白（HDL）胆固醇摩尔酯化速率（MERHDI）（P〈0．001），HDL胆固醇分数酯化速率FERHDL、Preβ．HDL（P〈0．01）等呈显著正相关，但与HDL及其亚类胆固醇无相关性（P〉0．05）。结论本研究建立了血清-巨噬细胞间胆固醇净流动的细胞分析体系和HPLC准确测定净流动的方法，为进一步研究血清．巨噬细胞间胆固醇净流动、HDL功能评价和心血管病危险分析提供了方法学基础。Objective To develop a safe and reliable method for the measurement of cholesterol net flux between macrophage cells and human serum by high performance liquid chromatography, and to investigate the relationship between cholesterol net flux and traditional risk factors of atheroselerosis. Methods J774 （murine cell line ） cells were plated in growth medium containing 5% individual human serum and cell cholesterol before and after incubation were measured by HPLC. Serum induced cell cholesterol net influx was calculated. Fasting blood samples of 75 apparently healthy volunteers （38 males and 37 females） from Oct to Dec 2010 were collected. Cholesterol net flux was measured by the newly established method. Spearman correlation analysis was used to evaluate the relationship between cholesterol net flux and lipid, lipoprotein, high-sensitivity C-reactive protein （hsCRP）and other CVD risk factors. Results When J774 cells were incubated with individual serum, cell cholesterol levels were elevated with the increase of serum concentrations and incubation time. J774 cells were incubated with medium containing 5% serum for 8 hours, the within assay and total CVs for the net movement of cellular cholesterol measured by HPLC were 0. 81% - 4. 03% and 4. 25% - 6. 58% , respectively. The average cholesterol net efflux of 75 healthy subjects was 10. 27 μg/mg, with a standard deviation of 2. 69 μg/mg protein, and a median of 10. 12 p,g/mgprotein. Cholesterol net efflux was significantly positively correlated with total cholesterol （ TC ）, triglycerides （ TG）, hsCRP, apolipoprotein （ apo ） CⅡ, apoC Ⅲ, small-dense lowdensity lipoprotein cholesterol （ LDLb-C ）, molar esterification rate of HDL cholesterol （ MERHDL ） （ P 〈 0. 001 ） ,fractional esterification rate of HDL cholesterol （ FERnDL） and Preβ-HDL（P 〈 0.01 ）. Cholesterol net efflux was not related with HDL as well as its subfractions. Conclusions A new method for the measurement of the net movement of cellular cholesterol b

关 键 词：巨噬细胞 胆固醇 色谱法 高压液相 动脉粥样硬化 

分 类 号：R282.71[医药卫生—中药学]