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目的探讨铜蓝蛋白(Cp)在石英诱导的人胚肺成纤维细胞(HELF)中JNK/ERK/AP-1信号通路改变中的作用。方法分别在100μg/ml石英作用前1 h、同时和作用后1h加入30μg/ml Cp,同时设立单独用石英和单独用Cp处理以及不做任何处理的细胞组,刺激细胞24 h后,用噻唑蓝(MTT)比色法观察Cp对石英诱导的HELF细胞增殖的影响。用100μg/ml石英分别作用于HELF细胞、转染JNK显性失活突变体质粒的HELF细胞(DN-JNK)和转染ERK显性失活突变体质粒的HELF细胞(DN-ERK)24 h;用100μg/ml石英作用1 h后,加入30μg/ml Cp作用24 h;同时设立不做任何处理的对照组。用MTT法检测分别抑制JNK和ERK后对细胞增殖的影响;用蛋白免疫印迹(Western blotting)实验检测JNK、ERK、c-Jun、c-Fos及其磷酸化水平的改变。结果石英作用1 h后加入Cp,对石英诱导的细胞增殖有促进作用,后续的实验选择这种作用模式。Cp可以明显增加JNK、ERK蛋白及磷酸化ERK(pERK)、磷酸化JNK(p-JNK)、磷酸化c-Jun(p-c-Jun)和磷酸化c-Fos(p-c-Fos)蛋白水平。抑制JNK蛋白活性后,Cp诱导的p-JNK、p-c-Jun和p-c-Fos蛋白水平的增加被抑制,Cp、石英以及Cp和石英共同诱导的细胞增殖加快被抑制;抑制ERK蛋白后,Cp诱导的p-ERK和p-c-Fos的增加被抑制,Cp、石英以及Cp和石英共同诱导的细胞增殖加快被抑制。结论 Cp通过JNK/c-Jun和c-Fos及ERK/c-Fos通路进一步增强石英诱导的促细胞增殖作用。
Objective To investigate the role of ceruloplasmin (Cp) in the JNK / ERK / AP-1 signaling pathway in quartz-induced human embryonic lung fibroblasts (HELF). Methods The cells were treated with quartz at a concentration of 1 μg / ml for 1 h before and 1 h after the addition of 1 μg / ml quartz respectively. The cells were stimulated with Cp for 24 h and treated with thiazole The effect of Cp on quartz induced proliferation of HELF cells was observed by MTT assay. HELF cells transfected with JNK dominant negative mutant plasmid (DN-JNK) and HELF cells transfected with ERK dominant negative mutant plasmid (DN-ERK) were treated with 100 μg / ml quartz for 24 h ; After treated with 100μg / ml quartz for 1 h, 30μg / ml Cp was added for 24 h; meanwhile, a control group without any treatment was established. The effects of inhibiting JNK and ERK on cell proliferation were detected by MTT assay. The changes of JNK, ERK, c-Jun, c-Fos and their phosphorylation were detected by Western blotting. Results Cp was added after quartz was added for 1 h, which promoted quartz cell proliferation. Follow-up experiments chose this mode of action. Cp could significantly increase the protein levels of JNK, ERK, pERK, p-JNK, p-c-Jun and p-cos-Fos. Inhibition of JNK protein activity, Cp-induced increase of p-JNK, pc-Jun and pc-Fos protein levels were inhibited, Cp, quartz and Cp and quartz co-induced accelerated cell proliferation was inhibited; inhibition of ERK protein, Cp induction The increase of p-ERK and pc-Fos was inhibited, and the acceleration of cell proliferation induced by Cp, quartz and Cp and quartz was inhibited. CONCLUSION Cp can further enhance quartz-induced pro-cellular proliferation via JNK / c-Jun and c-Fos and ERK / c-Fos pathways.