载脂蛋白E在ABCA1和ABCG1介导的胆固醇流出中的作用

Role of apolipoprotein E in cholesterol efflux mediated by ABCA1 and ABCG1

目的:探讨载脂蛋白E(ApoE)在ATP结合盒转运蛋白A1(ABCA1)和ATP结合盒转运蛋白G1(ABCG1)介导的胆固醇流出中的作用及可能机制。方法:将RAW 264.7细胞随机分为3组:空白对照组:用单纯培养基培养;低密度脂蛋白受体基因敲除(LDLr-/-)组:用20 mg/L LDLr-/-小鼠脂蛋白处理细胞;ApoE基因敲除(ApoE-/-)组:用20 mg/L ApoE-/-小鼠脂蛋白处理细胞。采用透射电镜和酶法检测细胞内脂质含量,用液闪仪技术检测胆固醇流出变化,用免疫印迹技术和实时定量PCR技术检测ABCA1和ABCG1的表达水平。结果:与对照组相比,LDLr-/-小鼠脂蛋白处理后细胞内脂质含量并未明显改变,而ApoE-/-小鼠脂蛋白处理48 h后,细胞内胆固醇酯增加了60%;同时,与LDLr-/-小鼠脂蛋白处理组相比,用ApoE-/-小鼠脂蛋白处理后,巨噬细胞胆固醇流出至载脂蛋白A-I(ApoA-I)和高密度脂蛋白(HDL)的量均明显降低。与对照组相比,LDLr-/-小鼠脂蛋白和ApoE-/-小鼠脂蛋白处理显著增加ABCA1和ABCG1的蛋白和mRNA水平,但是ApoE-/-小鼠脂蛋白对ABCA1和ABCG1蛋白和mRNA水平的诱导程度明显低于LDLr-/-小鼠脂蛋白。结论:ApoE在介导巨噬细胞胆固醇流出中发挥重要作用,该作用与其调节ABCA1和ABCG1的表达水平有关。

To investigate the role of apolipoprotein E （ApoE） in cholesterol efflux mediated by ATP- binding cassette transporter A1 （ ABCA1 ） and ATP - binding cassette transporter G1 （ABCG1）. METHODS ： RAW 264.7 cells were seeded in either 6 - well or 24 - well plates, and then incubated with 20 mg/L low - density lipoprotein receptor gene knockout （LDLr-/- ） mouse lipoprotein 20 mg/L ApoE gene knockout （ApoE-/- ） mouse lipoprotein or cul- ture medium alone. The changes of intracellular lipid content were measured by transmission electron microscopy and enzy- matic colorimetric method. The cholesterol efflux was determined by liquid scintillation. The mRNA and protein levels of ABCA1 and ABCG1 were detected by real - time PCR and Western blotting, respectively. RESULTS： The ApoE-/- mouse lipoprotein increased the content of intracellular cholesterol ester by 60% compared with the control cells. In addi- tion, ApoE-/- mouse lipoprotein treatment decreased the cholesterol efflux to apolipoprotein A- I （ApoA -I） and high- density lipoprotein （HDL） compared with LDLr-/- mouse lipoprotein treatment. ApoE-/- mouse lipoprotein treatment in- hibited the mRNA and protein levels of ABCA1 and ABCG1 compared with LDLr -/- mouse hpoprotein treatment. CON- CLUSION： Apolipoprotein E plays an important role in the cholesterol efflux of macrophages, which is associated with its regulatory effect on the expression of ABCA1 and ABCG1.