巨噬细胞ABCG1介导的氧化固醇外流的研究

Study on ABCG1-mediated oxysterol efflux from macrophage

目的测定巨噬细胞ABCG1介导的氧化固醇外流情况。方法利用ABCG1基因小干扰RNA慢病毒表达载体感染THP-1细胞,经有限稀释法筛选出敲低单克隆稳转株;用PMA诱导THP-1为巨噬细胞,ox-LDL(50μg/ml)刺激24 h后,通过液相色谱串联质谱测定细胞氧化固醇7β-羟基胆固醇(7β-OHC)和7-酮基胆固醇(7-KC)外流情况。结果对待测样品进行了氧化固醇定性与定量分析,发现与无外流接受体的空白对照组相比,诱导氧化型胆固醇外流率增加(7-KC:28.6%±2.3%比4.1%±0.7%,P<0.001;7β-OHC:35.5%±5.7%比3.7%±1.1%,P<0.001)。与阴性对照组相比,ABCG1敲低组氧化型胆固醇的外流率降低(7-KC:16.1%±3.1%比24.7%±2.3%,P<0.05;7β-OHC:15.7%±0.7%比33.1%±3.7%,P<0.001)。胞内氧化固醇沉积量通过细胞总蛋白进行校正,结果显示,与对照组比较,ABCG1低表达巨噬细胞胞内氧化固醇沉积增加[7-KC:(6.61±1.28)μg/mg比(2.13±0.08)μg/mg,P<0.05;7β-OHC:(1.61±0.51)μg/mg比(0.65±0.20)μg/mg,P<0.05]。结论巨噬细胞ABCG1低表达可抑制氧化固醇7β-OHC及7-KC外流,增加胞内氧化固醇沉积。

Objective To determine the macrophage ABCG1-mediated oxysterol efflux.Methods The THP-1 cells were infected with lentiviral vector of ABCG1 short hairpin RNA and the stable cell line was selected by limiting dilution method.PMA induced THP-1 macrophages was stimulated for 24 h with ox-LDL(50μg/ml),the outflow of oxidaized sterol 7β-hydroxycholesterol(7β-OHC)and 7-ketochlesterol(7-KC)efflux was measured by liquid chromatography tandem mass spectrometry.Results Qualitative and quantitative analysis of oxidized sterols was performed on the samples to be tested.Compared with the blank control group without efflux receptors,the induced oxidized cholesterol efflux rate significantly increased(7-KC:28.6%±2.3%vs.4.1%±0.7%,P<0.001;7β-OHC:35.5%±5.7%vs.3.7%±1.1%,P<0.001).Compared with the negative control group,the outflow rate of oxidized cholesterol in the ABCG1 knockdown group was significantly reduced(7-KC:16.1%±3.1%vs.24.7%±2.3%,P<0.05;7β-OHC:15.7%±0.7%vs.33.1%±3.7%,P<0.001).The amount of intracellular oxidized sterol deposition was corrected by total cell protein.The results showed that compared with the control group,the intracellular oxidized sterol deposition of macrophages with low ABCG1 expression increased significantly[7-KC:(6.61±1.28)μg/mg vs.(2.13±0.08)μg/mg,P<0.05;7β-OHC:(1.61±0.51)μg/mg vs.(0.65±0.20)μg/mg,P<0.05].Conclusions Low expression of macrophage ABCG1 can inhibit the efflux of 7β-OHC and 7-KC,and increase intracellular oxidized sterol deposition.