荧光标记胆固醇用于测定人单核巨噬细胞脂质外流

目的利用荧光标记的胆固醇(NBD-胆固醇)建立测定人巨噬细胞胆固醇外流的新方法,并与传统同位素标记胆固醇的测定方法进行比较。方法提取受试者外周血单核细胞分化为人巨噬细胞。体外培养人单核细胞系THP-1及鼠单核细胞系J774。以不同浓度的NBD-胆固醇孵育THP-1细胞,观察胆固醇浓度和孵育时间对细胞摄取的影响。用荧光酶标仪测定荧光信号强度以反映胆固醇摄取和外流。分别在三种细胞中测量NBD-胆固醇外流率,并在原代人巨噬细胞中观察与同位素标记胆固醇外流率的相关性。结果 NBD-胆固醇孵育细胞后迅速分布于细胞器中,但不进入细胞核。细胞对NBD-胆固醇的摄取有浓度依赖性,但达峰时间不受浓度影响。NBD-胆固醇外流率在各种巨噬细胞中呈现相似趋势,由载脂蛋白A1和高密度脂蛋白诱导的外流率在诱导外流4 h达到平台期。3H-胆固醇外流率在人巨噬细胞中与NBD-胆固醇外流率显著相关(r=0.968,P<0.001)。结论 NBD-胆固醇是一种有效的、敏感且环保的探针,可替代同位素标记的胆固醇用于巨噬细胞外流率的测定。

A New Assay of Cholesterol Efflux Rate in Human Peripheral Blood Monocyte Cells by Fluorescent Labeled Cholesterol

Aim In this study,fluorescence labeled cholesterol(NBD-cholesterol) was used to establish a new method to evaluate the cholesterol efflux,and compared NBD-cholesterol with 3H-cholesterol in assay of cholesterol efflux. Methods Isolated human peripheral blood monocyte cells from volunteers were differentiated into human macrophages.Human monocyte line THP-1 and mouse monocyte line J774 cells were cultured in vitro.THP-1 macrophages were incubated with different concentrations of NBD-cholesterol to choose an optimistic concentration and incubation time.NBD-cholesterol efflux rate was evaluated from three different kinds of macrophages.NBD-cholesterol uptake and efflux rate were reflected by fluorescent intensity measurement by multi-label counter.The NBD-cholesterol efflux capacity in human macrophages was estimated by the correlation to 3H-cholesterol efflux in human macrophages.Results NBD-cholesterol distributed rapidly into cells organelles except nucleus.Uptake of NBD-cholesterol in THP-1 macrophages was concentration dependent,but time to plateau was not dependent on concentration.NBD-cholesterol efflux rate reflected an appropriate cholesterol efflux rate in human macrophages,THP-1 macrophages and mouse macrophages.Both apolipoprotein A1 mediated and HDL mediated 3H-cholesterol efflux rates were significant correlated to NBD-cholesterol efflux rate in human macrophages(r=0.968,P<0.001).Conclusion NBD-cholesterol was a useful,sensitive and environmental protective probe for assay of human macrophage cholesterol efflux rate.