钙敏感受体对巨噬细胞泡沫化过程的影响及机制研究

Effect of Calcium-sensing Receptor on Human Macrophages Converting to Foam Cells With its Mechanisms

目的:研究钙敏感受体(CaR)在巨噬细胞中的表达情况及对巨噬细胞转化为泡沫细胞的影响。方法 :通过免疫荧光和蛋白印迹(Western Blot)检测巨噬细胞中CaR的表达情况;氧化低密度脂蛋白(ox-LDL)100 mg/L作用48 h,使巨噬细胞转化为泡沫细胞;实验分组为CaR激动剂组、CaR抑制剂组、空白对照组,分别作用泡沫细胞48 h:采用油红O染色检测阳性细胞数量;高效液相色谱观察细胞内胆固醇代谢情况;酶联免疫吸附法(ELISA)检测细胞上清液中炎性因子的分泌情况;Western blot检测细胞内CD36和胆固醇酰基转移酶(ACAT-1)蛋白的表达情况。结果 :Western blot和免疫荧光结果显示在巨噬细胞中存在CaR的表达。油红O染色和高效液相色谱检测泡沫细胞的结果显示:与空白对照组比较,CaR激动剂组阳性细胞数量和胆固醇酯、游离胆固醇、总胆固醇的含量均明显下降(P<0.05),而CaR抑制剂组阳性细胞数量和胆固醇酯、游离胆固醇、总胆固醇的含量均明显升高(P<0.05);ELISA检测结果显示:与空白对照组比较,CaR激动剂组促炎因子肿瘤坏死因子-(TNF-a)、巨噬细胞游走抑制因子(MIF)含量明显降低(P<0.05),抗炎因子白细胞介素-10(IL-10)含量明显升高(P<0.05),而CaR抑制剂组促炎因子TNF-a、MIF含量明显升高(P<0.05),抗炎因子IL-10含量明显降低(P<0.05);Western blot检测各组泡沫细胞结果发现:与空白对照组比较,CaR激动剂组CD36和ACAT-1蛋白表达明显下降,而CaR抑制剂组CD36和ACAT-1蛋白表达明显升高。结论 :在巨噬细胞中存在CaR的表达,并且高表达的CaR可以抑制巨噬细胞泡沫化的形成。

Objective： To study the expression of calcium-sensing receptor （CaR） in human macrophages and the effect of CaR on macrophages converting to foam cells with its mechanism. Methods： Human macrophages were treated with ox-LDL （100 mg/L） for 48 h to induce foam cell formation, the experimental foam cells were cultured in 3 groups for 48 h respectively. Blank control group, Foam cell ＋ CaR agonists （Agonists） group and Foam cell ＋ CaR inhibitor （Inhibitor） group. The CaR expression in macrophages was measured by immuno-fluorescence and Western blot analysis. The positive foam cell formation was detected by oil red O staining, the intracellular cholesterol metabolism in foam cell was observed by HPLC, the inlfammatory cytokine secretion in foam cell culture supernatant was examined by ELISA, the protein expressions of CD36 and acyl-coA： cholesterol acyl-transferase （ACAT-1） in foam cells was measured by Western blot analysis. Results： Immuno-lfuorescence and Western blot indicated CaR is expressed in human macrophages. Compared with Control group, Oil Red O staining and HPLC showed that Agonists group had less positive foam cell formation, decreased CE, FC , TC, and Inhibitor group had more positive foam cell formation, increased CE, FC, TC, allP〈0.05; ELISA presented that Agonists group had decreased TNF-α, MIF, increased anti-inflammatory cytokine IL-10, and Inhibitor group had increased TNF-α, MIF, decreased IL-10, allP〈0.05; Western blot analysis indicated that Agonists group had decreased protein expressions of CD36, ACAT-1 in foam cells, and Inhibitor group had increased protein expressions of CD36 and ACAT-1. Conclusion： CaR is expressed in human macrophages, the higher CaR expression may inhibit macrophages converting to foam cells.