骨髓间充质干细胞抑制巨噬细胞信号通路在缓解染硅尘小鼠肺泡炎中的作用
Inhibition of macrophage signaling pathway by bone marrow mesenchymal stem cells (BMSC) and alleviation of pulmonary alveolitis in mice exposed to silica dust
-
摘要:目的探讨骨髓间充质干细胞(BMSC)缓解染硅尘小鼠肺泡炎的机制。方法取无特定病原体级健康雄性C57BL/6小鼠10只, 选择5只小鼠以全骨髓贴壁法分离培养BMSC, 另外5只小鼠用于诱导骨髓巨噬细胞(BMDM)。取同类小鼠随机分为对照组、二氧化硅(SiO2)组以及SiO2染毒后BMSC移植组, 每组10只。采用气管暴露法, 将对照组小鼠经气管软骨间隙给予一次性注射0.90%氯化钠溶液20.0 μL; SiO2组和BMSC移植组小鼠先予一次性注射质量浓度为250 g/L的SiO2混悬液20.0 μL, 6 h后经鼠尾静脉分别一次性输注500.0 μL的0.90%氯化钠溶液或BMSC (细胞密度为1×109/L)。造模后第3 d处死小鼠, 免疫印迹检测小鼠肺组织中嗜中性白细胞碱性磷酸酶-3(NALP3)等炎性小体组分含量。Transwell体外共培养体系, 上室接种小鼠BMDM, 下室接种BMSC, 使用SiO2刺激BMDM, 酶联免疫吸附实验(ELISA)检测BMDM培养上清中白细胞介素(IL)-1β的表达情况, 免疫印迹检测BMDM中NALP3炎性小体组分含量。结果与SiO2组小鼠比较, BMSC移植组小鼠肺组织中白介素-1β前体(pro-IL-1β)、白介素-1β(IL-1β)、天冬氨酸蛋白水解酶-1前体(pro-caspase-1)、天冬氨酸蛋白水解酶-1(caspase-1)表达水平下调(P < 0.01)。BMSC共培养组BMDM培养上清中IL-1β浓度均低于对照组(P < 0.05), BMDM中pro-IL-1β、IL-1β、pro-caspase-1、caspase-1表达水平均低于SiO2组(P < 0.01), 直接加入不含细胞的BMSC培养上清(SN), 则上述指标无明显变化。结论BMSC可能抑制SiO2染尘小鼠的BMDM的NALP3炎性小体通路。Abstract:ObjectiveTo study the mechanism of bone marrow mesenchymal stem cell (BMSC)-mediated alleviation of pulmonary alveolitis in mice exposed to silica dust.MethodsThirty mice were randomly divided into 3 groups:control group, and two silica groups with or without BMSCs transplantation.Through the tracheal tube clearance, mice in control group received a single injection 20.0 μL of 0.90% sodium chloride solution by one time.Mice from in silica group and silica/BMSCs transplantation group first received a single injection of 20.0 μL silica dust suspension (mass concentration 250 g/L); followed by either 500.0 μL of 0.90% sodium chloride solution or by 500.0 μL of BMSCs suspension (cell density 1×109/L) through tail vein infusion 6 hours later.Mice were euthanized on the 3th day of the experiments.The levels of NALP3 inflammasome in lungs was determined by Western blot.Transwell system was used for co-culture of BMDM (in upper-chamber) and BMSC (in lower-chamber) co-culture.The level of cytokines IL-1β in BMDM cultural supernatant was detected by enzyme linked immunosorbent assay after stimulated by SiO2 stimulation.The levels of NALP3 inflammasome of in BMDM was determined by Western blot.ResultsThe levels of pro-IL-1β, IL-1β, pro-caspase-1, caspase-1 in lungs of silica/BMSCs transplantation group were lower than that in silica group (P < 0.01).In the experiment in vitro, the concentrations of IL-1β in SiO2 exposed BMSC/BMDM co-culture group were lower than the SiO2 exposure only groups (P < 0.05).Meanwhile, the levels of pro-IL-1β, IL-1β, pro-caspase-1, caspase-1 in BMDM was lower than that in silica group (P < 0.01).The level of these proteins didn′t change while when the cell-free supernatant of BMSC culture was directly added.ConclusionThe BMSC could inhibit NALP3 inflammasome of macrophages stimulated by SiO2.
下载: