载脂蛋白E在ABCA1和ABCG1介导的胆固醇流出中的作用

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RAW264.7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20mg/LLDLr^-/-小鼠脂蛋白处理细胞;ApoE基因敲除(ApoE^-/-)组:用20mg/LApoE^-/-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48h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。

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

AIM: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 culture medium alone.The changes of intracellular lipid content were measured by transmission electron microscopy and enzymatic 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 addition,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 inhibited the mRNA and protein levels of ABCA1 and ABCG1 compared with LDLr-/-mouse lipoprotein treatment.CONCLUSION: 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.