骨髓间充质干细胞对染矽尘小鼠肺部早期炎症抑制作用

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。

Silicosis

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.