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目的研究巨噬细胞冠蛋白1(coronin-1)与分枝菌酸(MA)诱导巨噬细胞泡沫化的相关性及其可能机制。方法根据冠蛋白1表达水平的差异,实验分为RAW264.7-Cor.Plus、RAW264.7和RAW264.7-Cor.Minus三组。各组细胞经100μg/m L MA包被的聚苯乙烯微球作用24 h后提取总蛋白,用Western blot法检测细胞冠蛋白1的表达水平。分别用(0、25、50、75、100)μg/m L MA包被聚苯乙烯微球作为吞噬颗粒,在2μmol/L松胞菌素D(cty D)处理前、后,分别吞噬24 h~8 d,用总胆固醇(TC)酶法测定试剂盒、游离胆固醇(FC)酶法测定试剂盒分别检测各组细胞TC、FC,再通过胆固醇酯(CE)和CE/TC比值定量评估巨噬细胞泡沫化水平。将cty D处理细胞(RAW264.7-cty D、RAW264.7-cty D-MA)及其对照组分别用异硫氰酸荧光素标记的鬼笔环肽(FITC-phalloidin)染色,计算纤维型肌动蛋白(F-actin)重排率。结果各组细胞受MA诱导后,其冠蛋白1的表达量显著高于相应对照组,其表达量从高至低依次为RAW264.7-Cor.Plus、RAW264.7、RAW264.7-Cor.Minus。表达不同水平冠蛋白1的各组细胞的泡沫化水平与MA包被剂量和MA作用时间呈正比;冠蛋白1表达量最高的RAW264.7-Cor.Plus细胞泡沫化水平最高,冠蛋白1表达量最低的RAW264.7-Cor.Minus细胞泡沫化水平最低;cty D抑制巨噬细胞F-actin显著降低MA诱导的细胞泡沫化水平,但抑制F-actin对冠蛋白1与MA诱导细胞泡沫化的正相关性并无显著影响;经鬼笔环肽染色后,MA诱导组细胞的F-actin重排率显著高于非MA诱导的对照组细胞。结论 MA可诱导巨噬细胞冠蛋白1的表达,其表达水平与MA诱导的细胞泡沫化呈正相关,F-actin参与MA诱导的细胞泡沫化过程,但F-actin并非冠蛋白1调控MA诱导细胞泡沫化的关键和唯一途径。
Objective To investigate the correlation between coronin-1 and mycolic acid (MA) -induced macrophage foam and its possible mechanism. Methods According to the difference of expression level of Crest 1, the experiment was divided into three groups: RAW264.7-Cor.Plus, RAW264.7 and RAW264.7-Cor.Minus. After the cells were treated with 100 microg / ml MA coated polystyrene microspheres for 24 hours, the total protein was extracted and the expression level of CGRP was detected by Western blot. Polystyrene microspheres were coated with (0, 25, 50, 75, 100) μg / mL MA as phagocytosis granules respectively before and after 2 μmol / L cty D treatment h to 8 days, the TC and FC of each group were detected by the enzymatic assay kit of total cholesterol (TC) and the kit of free cholesterol (FC), and then quantitatively evaluated by the ratio of cholesterol ester (CE) and CE / TC Macrophage foam level. The cty D-treated cells (RAW264.7-cty D, RAW264.7-cty D-MA) and their control groups were stained with fluorescein isothiocyanate-labeled FITC-phalloidin, Actin (F-actin) rearrangement rate. Results After the cells were induced by MA, the expression of CGRP in each group was significantly higher than that in the corresponding control group. The expression levels of the two groups were RAW264.7-Cor.Plus, RAW264.7 and RAW264.7-Cor. Minus. The foam level of each group of cells expressing different levels of canopy protein 1 was directly proportional to the MA coating dose and the time of MA action. The highest expression level of canopy protein 1 was present in RAW264.7-Cor.Plus cells, and the highest expression of canopy protein 1 Foam of RAW264.7-Cor.Minus cells with the lowest level was the lowest. F-actin of macrophage cty D significantly reduced the level of foam cells induced by MA but inhibited F-actin-induced cell foam There was no significant difference in the positive correlation between the F-actin rearrangement and the MA-induced cells. Conclusions MA can induce the expression of macrophage C-1, the expression level of C-MA was positively correlated with MA-induced cell-foaming and F-actin was involved in the MA-induced cell foaming process. Bubble is the key and the only way.