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本文旨在研究在鼠源巨噬细胞泡沫化过程中氧化低密度脂蛋白(oxidized low density lipoprotein,ox-LDL)对巨噬细胞内质网应激(endoplasmic reticulum stress,ERS)的诱导作用及其机制。体外培养RAW264.7巨噬细胞,分别给予ox-LDL(25、50和100mg/L)、抗CD36抗体+ox-LDL和衣霉素(tunicamycin,TM)等不同处理。采用油红O染色观察细胞内脂质蓄积情况,酶比色法测定细胞内总胆固醇含量,免疫细胞化学法检测ERS标志分子糖调节蛋白94(glucose-regulated protein94,GRP94)表达,免疫印迹法检测GRP94及未折叠蛋白反应关键分子p-IRE1(phosphorylated inositol-requiring enzyme1)和X盒结合蛋白1(X box binding protein1,XBP1)蛋白表达水平。结果显示,不同浓度(25、50和100mg/L)ox-LDL处理细胞24h后,胞浆内可见大量油红O染色阳性脂质颗粒,细胞内总胆固醇含量明显增加,分别为空白对照组的2.1倍、2.8倍和3.1倍;使用抗CD36抗体阻断ox-LDL的摄入,可显著减少100mg/Lox-LDL所致的细胞内胆固醇蓄积。不同浓度ox-LDL和ERS诱导剂TM均可显著增加GRP94及其上游信号分子p-IRE1和XBP1蛋白表达,且表达强度随着ox-LDL诱导浓度的增加而增强;抗CD36抗体显著抑制100mg/Lox-LDL所致的上述3种蛋白表达上调。上述结果提示,ox-LDL可呈剂量依赖性诱导RAW264.7巨噬细胞产生ERS,激活未折叠蛋白反应信号通路;该过程可能由清道夫受体CD36所介导。
The aim of this study was to investigate the effect of oxidized low density lipoprotein (ox-LDL) on the macrophage endoplasmic reticulum stress (ERS) in the process of murine macrophage foaming mechanism. RAW264.7 macrophages were cultured in vitro and treated with ox-LDL (25, 50 and 100 mg / L), anti-CD36 antibody + ox-LDL and tunicamycin (TM) respectively. The intracellular lipid accumulation was observed by oil red O staining, the intracellular total cholesterol was determined by enzyme colorimetric assay, the expression of ERS marker GRP94 was detected by immunocytochemistry, GRP94 and unfolded protein reaction key molecules p-IRE1 (phosphorylated inositol-requiring enzyme1) and X box binding protein1 (XBP1) protein expression levels. The results showed that a large number of oil-red O-stained liposomes were observed in the cytoplasm at different concentrations of ox-LDL (25, 50 and 100 mg / L) for 24 h and the content of total cholesterol in the cells increased markedly 2.1-fold, 2.8-fold and 3.1-fold. Using anti-CD36 antibody to block the uptake of ox-LDL could significantly reduce the intracellular cholesterol accumulation caused by 100mg / Lox-LDL. The expression of GRP94 and its upstream signaling molecules p-IRE1 and XBP1 were significantly increased by different concentrations of ox-LDL and ERS inducer TM, and the expression intensity of ox-LDL increased with the increase of ox-LDL concentration. Anti-CD36 antibody significantly inhibited the expression of GRP94, Lox-LDL-induced upregulation of the three proteins. These results suggest that ox-LDL can induce RAW264.7 macrophages to produce ERS in a dose-dependent manner and activate unfolded protein response signal pathway, which may be mediated by scavenger receptor CD36.