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目的探讨二氧化硅(Si O2)刺激的人肺泡上皮细胞系A549细胞水通道蛋白4(AQP-4)的表达及意义。方法将A549细胞分为0.5、1.0、2.0、4.0、8.0 h染尘组和抑制剂组,各染尘组分别以质量浓度为50 mg/L的Si O2混悬液刺激相应时间,抑制剂组先以浓度为100μmol/L的AQP-4抑制剂TGN-020孵育2.0 h,再以质量浓度为50mg/L的Si O2混悬液刺激1.0 h,另设不予任何处理的对照组;采用实时荧光定量聚合酶链式反应(RT-PCR)法检测AQP-4 mRNA表达。同时,将A549细胞分为3.0、6.0、12.0、24.0 h染尘组和抑制剂组,各染尘组分别以质量浓度为50 mg/L的Si O2混悬液刺激相应时间,抑制剂组先以浓度为100μmol/L的TGN-020孵育2.0 h,再以质量浓度为50 mg/L的Si O2混悬液刺激6.0 h,另设不予任何处理的对照组;分别采用免疫印迹法和免疫细胞化学法检测AQP-4蛋白表达。结果 RT-PCR分析结果显示:与对照组比较,Si O2刺激后A549细胞AQP-4 mRNA相对表达水平呈现先升高后逐渐下降趋势,于刺激1.0 h后达到高峰值(P<0.05),于刺激8.0 h后趋于正常水平(P>0.05)。免疫印迹法和免疫细胞化学法分析结果均显示:与对照组比较,Si O2刺激后A549细胞AQP-4蛋白呈现先升高后逐渐下降趋势,于刺激6.0 h后达到高峰值(P<0.05),但于刺激24.0 h后仍未下降至正常水平(P<0.01)。抑制剂组AQP-4的mRNA相对表达水平和蛋白表达水平均低于Si O2刺激相同时间的染尘组(P<0.01)。结论 Si O2刺激使A549细胞AQP-4的表达增高,其特异性抑制剂可抑制Si O2刺激引起的AQP-4表达增高,推测AQP-4可能参与了矽肺的发生发展过程。
Objective To investigate the expression of aquaporin 4 (AQP-4) in human lung alveolar epithelial cell line A549 stimulated by silicon dioxide (Si 2 O) and its significance. Methods A549 cells were divided into two groups: control group (0.5,1.0,2.0,4.0 and 8.0 h) and inhibitor group (control group). In each group, Si02 suspension with mass concentration of 50 mg / L was used to stimulate the corresponding time. First, the cells were incubated with AQP-4 inhibitor TGN-020 at a concentration of 100 μmol / L for 2.0 h and then stimulated with Si O2 suspension at a concentration of 50 mg / L for 1.0 h. The control group was treated with real-time Fluorescent quantitative polymerase chain reaction (RT-PCR) was used to detect the expression of AQP-4 mRNA. At the same time, A549 cells were divided into 3.0, 6.0, 12.0 and 24.0 h dyed dust and inhibitor groups, and each dyed dust group was stimulated with Si O2 suspension at the concentration of 50 mg / L for the corresponding time. The cells were incubated with TGN-020 at a concentration of 100 μmol / L for 2.0 h and then stimulated with Si 2 O 5 at a concentration of 50 mg / L for 6.0 h. A control group without any treatment was also established. Immunoblotting and immunization Cytochemical detection of AQP-4 protein expression. Results Compared with the control group, the relative expression level of AQP-4 mRNA in A549 cells increased first and then decreased, and reached the peak value at 1.0 h (P <0.05), compared with the control group After stimulation for 8.0 h, it tended to be normal (P> 0.05). Immunocytochemistry and immunocytochemistry analysis showed that compared with the control group, AQP-4 protein of A549 cells increased first and then decreased gradually after Si O2 stimulation, and peaked at 6.0 h after stimulation (P <0.05) , But it did not decrease to normal level after 24.0 h stimulation (P <0.01). The relative expression level and protein expression level of AQP-4 in inhibitor group were lower than that in Si02-treated group (P <0.01). Conclusions Si O2 stimulation increases the expression of AQP-4 in A549 cells. The specific inhibitor of Si O2 can inhibit the increase of AQP-4 expression induced by Si O2 stimulation. It is speculated that AQP-4 may be involved in the development and progression of silicosis.