矽肺患者巨噬细胞培养上清通过p38MAPK对人胚肺成纤维细胞增殖的影响

Effect of p38MAPK on the proliferation in human embryonic lung fibroblasts in vitro

目的 探讨矽肺患者巨噬细胞（AM）培养上清是否通过激活p38丝裂原活化蛋白激酶（p38MAPK）信号转导通路促进人胚肺成纤维细胞（HELF）的增殖作用,进而参与矽肺纤维化的发生发展过程.方法 以支气管肺泡灌洗法收集矽肺患者AM,在体外用含有SiO2（50 μg/ml）的DMEM培养基和不含SiO2的DMEM培养基培养18h,然后收集培养的AM上清液.用组织贴块法获取培养HELF,与AM上清液共同孵育,将HELF分为空白对照组、AM组、SiO2＋AM组、SB203580＋SiO2＋AM组,用噻唑蓝（MTT）法和流式细胞仪法分别检测各组HELF的增殖情况和细胞周期.结果 SiO2＋AM组细胞的平均吸光度值为0.48±0.03,与空白对照组（0.29±0.01）、AM组（0.38±0.02）、SB203580＋SiO2＋AM组（0.33±0.03）的差异有统计学意义（P〈0.05）;SiO2＋AM组细胞增殖指数为18.12±0.82,与空白对照组（9.24±0.48）、AM组（14.76±0.43）、SB203580＋SiO2＋AM组（11.71±0.70）的差异有统计学意义（P〈0.05）.SB203580＋SiO2＋AM组的细胞吸光度值和细胞增殖指数均明显降低.结论 经SiO2刺激的矽肺患者AM培养上清通过激活p38MAPK信号转导通路可以促进HELF增殖.

Objective To study the proliferation effect of the AM supernatant incubated activation of p38 mitogen activated protein kinases （p38MAPK） signal transduction pathway in human embryonic lung fibroblasts,and to participate in the development of fibrosis in silicosis. Methods The silicotic alveolar macrophages were collected by bronchoalveolar lavage and incubated in vitro in the DMEM medium containing SiO2 （50 μg/ml） and DMEM medium without SiO2 for 18 h. Then the AM supernatant incubated for 18 h was collected. HELFs were isolated by organize paste block method, and incubated with AM supernatants. HELFs were divided into four groups：blank control groups, AM groups, SiO2＋AM groups, SB203580＋SiO2＋AM groups.The proliferation in the HELF was detected with MTT method and Flow cytometry. Results The proliferation in the HELF acted with the conditioned AM supernatant fluid were more than blank control groups, AM groups and SB203580 ＋ SiO2 ＋ AM groups （average optical density： 0.48±0.03 vs 0.29±0.01,0.38±0.02,0.33±0.03）,the values with MTT method were statistically different （P〈0.05）; Proliferous index with flow cytometry in SiO2＋ AM groups （ 18.12 ±0.82 ） was bigger than blank control groups （9.24±0.48）, AM groups （ 14.76±0.43 ） and SB203580＋SiO2＋AM groups （11.71 ±0.70） and the values were statistically different （P〈0.05）. Conclusions The AM supernatant stimulated by silicon dioxide can accelerate the proliferation in the HELF by activation of p38MAPK signal transduction pathway.