二氧化硅刺激A549细胞水通道蛋白-1表达:体外研究
Stimulative Effect of Silicon Dioxide on Expression of Aquaporin-1 in A549 Cells:An in vitro Study
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摘要:
目的 研究二氧化硅(SiO2)粉尘刺激对人肺泡上皮A549细胞水通道蛋白-1(AQP-1)的表达及意义。 方法 将A549细胞分为对照组、染毒组及抑制剂组。其中染毒组细胞利用50 mg/L的SiO2混悬液刺激细胞,SiO2分别刺激0.5、1、2、4、8 h后检测mRNA表达; SiO2分别刺激3、6、12、24 h后检测蛋白表达。抑制剂组则先加入AQP-1特异性的通道抑制剂氯化汞(HgCl2)孵育3 min后再加SiO2刺激2 h检测mRNA,刺激6 h检测蛋白表达。各组采用实时聚合酶链反应法检测AQP-1 mRNA表达水平,Western blot、免疫细胞化学检测AQP-1蛋白表达水平。 结果 AQP-1mRNA表达:与对照组相比,SiO2刺激后的0.5、1、2、4 h A549细胞AQP-1 mRNA的表达量分别是对照组的2.78、3.52、3.85、1.98倍,8 h时回到对照组水平(P<0.01);抑制剂组的表达水平为相同刺激时间SiO2染毒组的65%。AQP-1蛋白表达:①免疫印迹检测结果显示,与对照组相比,SiO2刺激后的3、6、12、24 h A549细胞的表达量分别为对照组的1.44、2.56、1.93、1.35倍(P<0.05或P<0.01);抑制剂组的表达水平低于相同刺激时间SiO2染毒组,为其70%(P<0.01)。②免疫细胞化学结果显示,与对照组相比,SiO2刺激后的3、6、12、24 h A549细胞的表达量分别为对照组的1.17、1.49、1.29、1.07倍(P<0.05或P<0.01)。抑制剂组的表达水平低于相同刺激时间SiO2染毒组,为其71%(P<0.01)。 结论 SiO2刺激使A549细胞的AQP-1表达增高,其特异性抑制剂氯化汞可抑制SiO2刺激引起的AQP-1的表达增高,推测AQP-1可能参与了矽肺的发生发展过程。 Abstract:Objective To study the expression and significance of aquaporin-1 (AQP-1) in human alveolar epithelial line A549 cells stimulated by silica. Methods A549 cells were divided into control, silica-stimulated, and inhibitor groups. The cells of the silica-stimulated group and the inhibitor group were stimulated by 50 mg/L silicon dioxide dispersed suspension to detect their mRNA expressions after 0.5, 1, 2, 4, and 8 h and protein expressions after 3, 6, 12, and 24 h. The cells of the inhibitor group were pretreated with mercuric chloride (specific channel inhibitor of AQP-1) for 3 minutes and then stimulated by silicon dioxide for 2 h to detect mRNA expression or for 6 h to detect protein expression. The expression of AQP-1 protein was detected by Western blot and immunocytochemistry and the expression of AQP-1 mRNA of the cells was detected by real-time polymerase chain reaction. Results Compared with the control group, the expression level of AQP-1 mRNA in silica-stimulated cells was 2.78, 3.52, 3.85, and 1.98 times after 0.5, 1, 2, and 4 h of silica-stimulation respectively (P<0.01), and returned to the control level after 8 h; the expression level of the inhibitor group was 65% of the corresponding silica-stimulated group. According to Western blot assay, the expression level of AQP-1 protein of the silica-stimulated cells was 1.44, 2.56, 1.93, and 1.35 times of the control group after 3, 6, 12, and 24 h of silica-stimulation respectively (P<0.05 or P<0.01); the level of the inhibitor group was 70% of the corresponding silica-stimulated group (P<0.01). According to immunocytochemistry, the expression level of AQP-1 protein was 1.17, 1.49, 1.29, and 1.07 times of the control group after silica administration for 3, 6, 12, and 24 h respectively (P<0.05 or P<0.01); that of the inhibitor group was 71% of the corresponding silica-stimulated group (P<0.01). Conclusion Elevated expression levels of AQP-1 mRNA and protein are induced by silica, and the specific inhibitor mercuric chloride could inhibit the increased expression, suggesting that AQP-1 participates the development of silicosis.
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