青石棉诱导BEAS-2B细胞ERK1/2磷酸化表达的研究

目的 了解青石棉在致癌过程中引起信号转导蛋白变化的特点.方法 使用人呼吸道上皮细胞株（human bronchial epithelial cell line,BEAS-2B）体外培养,以终浓度100 μg/ml的青石棉和100nmoL/L表皮生长因子（epidermal growth factor,EGF）分别刺激BEAS-2B细胞30和120min,使用特异性抗磷酸化细胞外调节蛋白激酶（extracellular regulated protein kinase,ERK1/2）、ERK激酶（ERK kinase,MEK1/2）和抗总ERK1/2、MEK1/2抗体进行Western免疫印迹,检测相应的蛋白表达水平.结果 青石棉刺激BEAS-2B细胞30 min后,可诱导磷酸化ERK1/2的快速高表达,且此高表达可持续至120 min,与对照组比较,差异有统计学意义（P＜0.05）;EGF作为诱导磷酸化ERK1/2的阳性刺激物,在30和120 min两时段内均可诱导ERK1/2的激活,与对照组比较,差异有统计学意义（P＜0.05）;任何时间段刺激BEAS-2B,在对照、青石棉和EGF组间,总ERK1/2表达差异无统计学意义（P＞0.05）.青石棉刺激BEAS-2B细胞30、120 min后,可诱导磷酸化MEK1/2的快速高表达,与对照组比较,差异有统计学意义（P＜0.05）.结论 青石棉可快速诱导BEAS细胞产生磷酸化ERK1/2、MEK1/2蛋白的高表达,提示MAPKs参与了青石棉所致疾病的过程.

Expression of phosphorylated ERK1/2 induced by crocidolite fibers in BEAS-2B cells

OBJECTIVE: To explore the characteristic of the signal transduction in BEAS cells induced by the crocidolite fibers. METHODS: The human respiratory airway epithelial cells BEAS-2B were cultured in vitro. The final 100 microg/ml crocidolite concentration and lOnM of epidermal growth factor were cocultured with BEAS-2B cells for 30 minutes and 120 minutes. Phosphorylated ERKl/2 and MEKl/2 were detected by Western Blotting using specific antibodies. RESULTS: A rapid phosphorylation expression of ERK1/2 (molecular weight at 44 kD and 42 kD, also called as p44 and p42) was observed by treatment of the BEAS-2B cells with 100 microg/ml crocidolite or 100 ng/ml EGF (the proven activator of the ERK signaling pathway) at 30 minutes. This phosphorylation could be still detected by incubation the cells at 2 hours. However no expression was changed for the total ERKl/2 expression at 30 minutes or 120 minutes. Treatment of BEAS cells with 100 microg/ml crocidolite fiber or 100 ng/ml EGF led to the rapid increased phosphorylation of MEK1/2 at 30 minutes; similarly, the overexpression of MEK1/2 could last 2 hours. CONCLUSION: The crocidolite induces the MAPK (ERK1/2 and MEK1/2) phosphorylation within a shorter time. It indicates that the MAPKs signals are involved in the process of crocidolite induced damage.