二氧化硅刺激A549细胞水通道蛋白4表达研究

发布时间：2018-09-12 19:32

【摘要】：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(P0.05),于刺激8.0 h后趋于正常水平(P0.05)。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(P0.05),但于刺激24.0 h后仍未下降至正常水平(P0.01)。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(P0.01)。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。
[Abstract]:Objective to investigate the expression and significance of aquaporin 4 (AQP-4) in human alveolar epithelial cell line A549 stimulated by silica (Si O 2). Methods A549 cells were divided into two groups: the group of 0. 5, 1. 0 and 2. 0 and the control group. Each group was stimulated with 50 mg/L Si O 2 suspension for the corresponding time. The inhibitor group was incubated with AQP-4 inhibitor TGN-020 (100 渭 mol/L) for 2.0 h, and then stimulated with Si O 2 suspension of 50mg/L for 1.0 h. The AQP-4 mRNA expression was detected by real-time fluorescence quantitative polymerase chain reaction (RT-PCR). At the same time, A549 cells were divided into 3. 0 0? 0? 0 and 24. 0 h exposure group and inhibitor group. Each group was stimulated with 50 mg/L Si O 2 suspension for the corresponding time. The inhibitor group was incubated with 100 渭 mol/L TGN-020 for 2.0 h, then stimulated with 50 mg/L Si O 2 suspension for 6.0 h. The control group was set up without any treatment, and the expression of AQP-4 protein was detected by Western blotting and immunocytochemistry, respectively. Results the results of RT-PCR analysis showed that the relative expression of AQP-4 mRNA in A549 cells increased at first and then decreased gradually compared with the control group, and reached a peak value at 1.0 h (P0.05) and a normal level at 8.0 h (P0.05). The results of immunoblotting and immunocytochemistry showed that compared with the control group, the AQP-4 protein of A549 cells increased firstly and then decreased gradually. The peak value was reached at 6.0 h after stimulation (P0.05), but it did not decrease to normal level at 24.0 h (P0.01). The relative mRNA expression and protein expression of AQP-4 in the inhibitor group were lower than those in the dust-exposed group at the same time after stimulation by Si O _ 2 (P0.01). Conclusion Si O 2 stimulation can increase the expression of AQP-4 in A549 cells, and its specific inhibitor can inhibit the increase of AQP-4 expression induced by Si O 2 stimulation. It is suggested that AQP-4 may be involved in the pathogenesis and development of silicosis.
【作者单位】：河北联合大学附属医院呼吸内科;河北联合大学临床医学院;河北联合大学医学实验研究中心;
【基金】：河北省科技厅科技支撑项目(10276116D)
【分类号】：R135.2

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