二氧化硅刺激A549细胞水通道蛋白4表达研究  

Effect of silicon dioxide on expression of aquaporin-4 in A549 cells

作　　者：喻昌利[1,2] 张芳[2] 郝小惠[3] 刘和亮[3] 

机构地区：[1]河北联合大学附属医院呼吸内科,河北唐山063000 [2]河北联合大学临床医学院,063000 [3]河北联合大学医学实验研究中心,063000

出　　处：《中国职业医学》2014年第6期642-647,共6页China Occupational Medicine

基　　金：河北省科技厅科技支撑项目(10276116D)

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(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 study the expression and significance of aquaporin-4 （AQP-4） in human alveolar epithelial line A549 cells stimulated by Silica. Methods A549 cells were divided into 7 groups ： the 0.5, 1.0, 2.0, 4.0 and 8.0 hoursilica-stimulated groups stimulated with sihca （mass concentration of 50 rag/L） in the corresponding time; the inhibitor group was pretreated with TGN-020 （ specific channel inhibitor of AQP-4, concentration of 100 trmol/L） for 2.0 hours and then stimulated with the same concentration of silica for h 0 hour; the control group was untreated. The expression of AQP-4 mRNA was detected by real-time fluorescent quantitative polymerase chain reaction （RT-PCR）. At the same time, A549 cells were divided into 6 groups ： the 3.0, 6.0, 12.0 and 24.0 hour-silica-stimulated groups were stimulated with silica （mass concentration of 50 mg/L） in the corresponding time; the inhibitor group was pretreated with TGN-020 （concentration of 100 μmol/L） for 2.0 hours and then stimulated with the same concentration of silica for 6.0 hours; the control group was untreated. Both of western blot and immunocytochemistry were used to detect the expression of AQP-4 protein. Results RT-PCR analysis showed that, compared with that of the control group, the relative expression of AQP-4 mRNA in the silica-stimulated groups increased first and then decreased as the silica stimulated time prolonged （ P 〈 0.05 ）, with the peak vale of relative expression of AQP-4 mRNA at 1.0 hour after silica stimulated （ P 〈 0.05 ） and returned to normal 8.0 hours after silica stimulated （P 〉 0.05）. Western blot and immunocytochemistry assay demonstrated that, compared with that of the control group, the expression of AQP-4 protein in the silica-stimulated groups increased first and then decreased as the silica stimulated time prolonged, with the peak vale of expression of AQP-4 protein at 6.0 hours after silica stimulated （P 〈0.05）, but it did not returned to the normal 24.0 hours after silica

关 键 词：水通道蛋白4 二氧化硅 A549细胞 矽肺 聚合酶链式反应 免疫印迹法 免疫细胞化学法 

分 类 号：R135.2[医药卫生—劳动卫生]