细胞外信号调节激酶通路对二氧化硅活化的肺成纤维细胞增殖的影响

Effect of ERK1/2 signal pathway on the proliferation of lung fibroblast activated by SiO2

目的 探讨细胞外信号调节激酶（ERK）信号转导通路对经SiO2活化的矽肺患者肺泡巨噬细胞（AM）条件上清刺激的人胚肺成纤维细胞（HELF）增殖的影响.方法 将矽肺患者支气管肺泡灌洗液中的AM体外再次染尘,取其上清与HELF共同孵育,用特异性阻断剂PD98059进行干预,实验分为空白对照组、AM对照组、SiO2处理组、PD98059干预组,通过噻唑蓝（MTT）法、流式细胞术法及免疫印迹（Western blot）法观察成纤维细胞细胞增殖活性及p-ERK1/2蛋白表达情况.结果 SiO2处理组及AM对照组细胞增殖的吸光度（A值）增高,分别是空白对照组的2.6和2.0倍,差异有统计学意义（P＜0.05）.25和50 μmol/L PD98059组HELF细胞增殖的A值分别是SiO2处理组的72.1%和48.5%,差异有统计学意义（P＜0.05）.经SiO2粉尘刺激的AM培养上清作用于HELF后,p-ERK1/2表达明显上调,15min时已有表达,30min时活化明显,表达最强（A值为0.4653±0.0265）,60min后仍处于较高的状态而表达有所减弱.除15 min外,SiO2处理组各时间点ERK1/2的蛋白表达分别是同期AM对照组的1.25、1.23、1.25倍,差异均有统计学意义（P＜0.05）.结论 加尘AM培养上清可能通过ERK1/2信号途径促进HELF的增殖及ERK1/2的活化,而参与矽肺的发生发展.

Objective To observe the effect of ERK 1/2 signal pathway activated by SiO2 in the proliferation of human embryonic lung fibroblast mediated by silicotic alveolar macrophages （AM）. Methods The AM harvested from silicotic sufferers by bronchoalveolar lavage （BAL） were interacted with SiO2 suspension once more. HELF, pretreated with the inhibitor PD98059（50 μmol/L） for 1 hour, were stimulated by conditional supernatant fluid of silicotic sufferers. The experimentation were divided into four groups： blank group, AM control group, SiO2 treatment group, PD98059 intervention group. The proliferation activity and expressions of phospho-ERK1/2 of lung fibroblast activated by AM supernatant fluids of silicotic were detected with the MTT assay, flow cytometry and Western blot method after being pretreated with PD98059. Results The A values of cell proliferation in SiO2 treatment group and AM control group were 2.6 and 2.0 times than that of blank group, in which the difference was statistically significant （P〈0.05=. Compared with SiO2 treatment group, the A values of every concentration of PD98059 intervention group decreased with a dose - response relationship, after 10, 25 and 50 μmol/L PD98059 intervention. The 25 and 50 μmol/L PD98059 intervention group were 72.1% and 48.5% of SiO2 treatment group, which the difference was statistic （P〈0.05=. The expression of phospho-ERK1/2 in SiO2 treatment group was up, which appeared in 15 min and apparent activated in 30 min （A value was 0.4653±0.0265）, and then still in the higher state afterwards declined after 60 min. In addition to 15 min, the expression of phospho-ERK1/2 protein in SiO2 treatment group at each time point were 1.25, 1.23,1.25 times over the same period AM control group respectively, the differences were statistically significant（P〈0.05 =.Conclusion The silicotic supernatant of AM promotes proliferation of HELF and activation of ERK1/2,which may involve in the development of silicosis pathogenesis by ERK1/2 signal pathway.