溶酶体在巨噬细胞极化中的作用研究

Role of lysosomes in macrophage polarization

目的探讨溶酶体在巨噬细胞极化中的作用。方法体外培养小鼠单核巨噬细胞系RAW264.7,用脂多糖(LPS)100 ng/ml+伽马干扰素(IFN-γ)20 ng/ml诱导其向经典活化型(M1)极化,用白细胞介素4(IL-4)20 ng/ml诱导其向替代活化型(M2)极化,建立巨噬细胞极化模型。以未诱导细胞作为对照组。通过流式细胞术、ELISA、Western blot及实时PCR(real-time PCR)法,检测M1型及M2型巨噬细胞比例及标志物表达,验证巨噬细胞极化模型是否建立成功。通过Western blot、real-time PCR及免疫荧光染色检测M1型及M2型巨噬细胞中溶酶体标志物表达,包括溶酶体膜相关蛋白1、2(LAMP-1、LAMP-2)及溶酶体整合膜蛋白2(LIMP-2)。给予溶酶体抑制剂氯喹(chloroquine)25μmol/L刺激巨噬细胞,流式细胞术检测M1、M2型巨噬细胞比例。结果成功建立巨噬细胞极化模型。与对照组比较,溶酶体标志物(LAMP-1、LAMP-2及LIMP-2)转录及蛋白水平表达在M1型极化组中降低,而在M2型极化组中增加。溶酶体抑制剂氯喹处理可降低M2型极化比例,增加M1型极化比例。结论溶酶体参与巨噬细胞极化过程,抑制溶酶体可促进巨噬细胞向M1型极化。

Objective To investigate the role of lysosomes in macrophage polarization. Methods In vitro model of macrophage polarization was established with RAW 264.7 cell line. M1 macrophages were obtained by co-incubating of cells with lipopolysaccharide （LPS, 100ng/ml） plus interferon-g （IFN-g, 20？ng/ml）. M2 macrophages were obtained by co-incubating of cells with interleukine-4 （IL-4, 20ng/ml）. Chloroquine （25？μmol/l）, a well-accepted lysosome inhibitor, was used to treat RAW 264.7 cells. RAW 264.7 cells in sham control group did not received any treatments. Flow cytometry, ELISA, Western blot and Real-time PCR were performed to measure differential success of two cell types and lysosome markers expression including lysosome associated membrane protein 1, 2 （LAMP-1, LAMP-2） and lysosome integral membrane protein 2 （LIMP-2）. Results Flow cytometry data suggested that in vitro model of macrophage polarization was successfully established. Compared with the control group, expression of lysosome makers including LAMP-1, LAPM-2 and LIMP-2 were signifcantly decreased in M1 macrophage subgroup while a dramatic increase of mentioned biomarkers of lysosomes was witnessed in M2 macrophages. In addition, treatment of chloroquine signifcantly decreased ratio of M2 macrophages over M1 macrophages. Conclusions Lysosome participates in macrophage polarization, and inhibition of lysosome facilitates differentiation of M1 polarization.