目的 探讨Sortilin对荷脂THP-1巨噬细胞ABCA1蛋白表达及胞内脂质流出的影响。 方法 采用Western blot和qRT-PCR检测细胞内ABCA1蛋白和mRNA表达水平；Co-IP实验检测Sortilin和ABCA1的结合情况；高效液相色谱法检测细胞内总胆固醇、游离胆固醇、胆固醇酯含量变化；油红O染色观察细胞内脂滴情况。 结果 Sortilin下调细胞内ABCA1蛋白水平，但对其mRNA水平无显著影响；Co-IP结果表明Sortilin能与ABCA1结合；与对照组相比，Sortilin过表达后荷脂巨噬细胞内胆固醇流出减少，胞内脂质含量增加且脂滴肥大，数量明显增多，Sortilin沉默后荷脂巨噬细胞胆固醇流出增加，胞内脂质含量减少，脂滴瘦小，数量减少；溶酶体抑制剂氯喹共处理可部分逆转Sortilin过表达对巨噬细胞ABCA1蛋白和胆固醇流出的抑制作用。 结论 Sortilin下调巨噬细胞ABCA1蛋白水平，抑制胆固醇流出，促进胞内脂质蓄积。
Abstract
Objective To investigate the effect of Sortilin on protein level of macrophage ABCA1 and intracellular lipid efflux. Methods The protein and mRNA levels of lipid-laden macrophage ABCA1 were detected by western blot (WB) and quantitative real-time polymerase chain reaction (qRT-PCR) respectively; the combination of Sortilin with ABCA1 was detected by co-immunoprecipitation (Co-IP); macrophage cholesterol efflux was measured by liquid scintillation counting. Total cholesterol (TC), free cholesterol (FC) and cholesterol ester (CE) were determined by high performance liquid chromatography (HPLC). Intracellular lipid droplets were observed by oil red O staining. Results Sortilin overexpression significantly decreased protein level of ABCA1, while Sortilin silence obviously raised cellular level of ABCA1 protein in lipid-laden macrophage. ABCA1 mRNA has no significant change in these three groups. Co-IP assay showed that Sortilin combined with ABCA1 protein. Compared to control group, Sortilin overexpression decreased cholesterol efflux from lipid-laden macrophage, resulting in macrophage cholesterol accumulation and lipid droplet generation. Sortilin silence dramatically enhanced cholesterol efflux and decreased lipid droplet formation. Co-treatment with chloroquine can partially abolish the inhibitory role of Sortilin overexpression in ABCA1 protein and lipid efflux of lipid-laden macrophage. Conclusions Sortilin reduces protein level of macrophage ABCA1 and intracellular cholesterol efflux, causes the accumulation of intracellular lipid.
关键词
Sortilin /
巨噬细胞 /
ABCA1 /
胆固醇流出
Key words
Sortilin /
Macrophage /
ABCA1 /
Cholesterol efflux
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基金
国家自然科学基金(81770460)；加拿大萨斯喀彻温省卫生研究基金会博士后基金(SHRF4144)；衡阳市科学技术发展计划项目（2017KJ268、2017KJ182）；南华大学博士启动基金(2014XQD37)；大学生研究性学习和创新性实验计划项目（201810555015）：湖南省自然科学基金（2020JJ4532)
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