背景：胆固醇与动脉粥样硬化等疾病的发生、发展密切相关。目前研究细胞胆固醇动态变化的方法有局限性。 目的：通过BODIPY-Cholesterol标记RAW 264.7小鼠巨噬细胞，检测细胞胆固醇的外流率，并研究细胞外胆固醇浓度和脂多糖对其的影响。 方法：RAW264.7细胞采用含有体积分数10%FBS的DMEM培养基体外培养，再用0.025 mmol/L的BODIPY-Cholesterol标记细胞1，2，4，8 h，以无血清DMEM洗涤细胞后，再孵育细胞6，12，24，48，96 h，优化标记细胞的时间及孵育时间。分别采用胆固醇、脂多糖、异常高胆固醇人血清和正常胆固醇人血清处理细胞，再对其胆固醇外流率进行测定和比较。 结果与结论：BODIPY-Cholesterol标记细胞2-8 h后，测定胆固醇外流率的结果较好。RAW 264.7小鼠巨噬细胞经过BODIPY-Cholesterol荧光染料标记2 h后，测定胆固醇外流率，发现其随外加胆固醇浓度(0.1，0.5，2.5 mmol/L)的增大呈递减趋势(P<0.01)。脂多糖刺激后细胞的胆固醇外流率降低(P<0.05)。采用高胆固醇人血清处理细胞后，胆固醇的外流率降低(P<0.05)。结果说明，利用BODIPY-Cholesterol可以定量测定细胞胆固醇外流率，并能研究环境中胆固醇和脂多糖对其的影响。%BACKGROUND:Cholesterol is closely linked to the occurrence and progression of atherosclerosis. Current approaches to study celular cholesterol dynamics have their own limitations. OBJECTIVE: To measure the cholesterol efflux rate of RAW 264.7 mouse macrophages by BODIPY-Cholesterol labeling and to explore the effects of extracelular cholesterol and lipopolysaccharide on the cholesterol efflux rate. METHODS:RAW 264.7 cels were cultured in vitro with DMEM containing 10% fetal bovine serum, and labeled with BODIPY-Cholesterol for 1, 2, 4, 8 hours. Then, the cels were rinsed with serum-free DMEM and inoculated for 6, 12, 24, 48, 96 hours to optimize the labeling time and incubation time. We measured and compared the cholesterol efflux rates after cultured cels were treated with cholesterol, lipopolysaccharide, human sera with high cholesterol or human sera with normal cholesterol. RESULTS AND CONCLUSION: The best labeling time for BODIPY-Cholesterol was 2-8 hours. Cholesterol efflux rates were gradualy decreased after the cels that were labeled for 2 hours were incubated with increasing concentrations of cholesterol (0.1, 0.5, 2.5 mmol/L,P< 0.01). Treating cels with lipopolysaccharide also decreased the cholesterol efflux rate (P < 0.05). Furthermore, the cholesterol efflux rate was decreased after cels were treated with human sera with high cholesterol (P< 0.05). These findings indicate that BODIPY-Cholesterol can be used to measure celular cholesterol efflux rate and to study the effects of extracelular cholesterol and lipopolysaccharide on the cholesterol efflux rate.
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