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[目的]研究二氧化硅(Si O2)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Si O2混悬液刺激细胞,Si O2分别刺激0.5、1、2、4、8 h后检测m RNA表达;Si O2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(Hg Cl2)孵育3 min后再加Si O2刺激2 h检测m RNA,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 m RNA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-1m RNA表达:与对照组相比,Si O2刺激后的0.5、1、2、4 h A549细胞AQP-1 m RNA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Si O2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,Si O2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Si O2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,Si O2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Si O2染毒组,为其71%(P<0.01)。[结论]Si O2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Si O2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。
[Objective] To investigate the expression and significance of aquaporin-1 (AQP-1) in human alveolar epithelial A549 cells stimulated with Si O2 dust. [Methods] A549 cells were divided into control group, exposure group and inhibitor group. The cells were stimulated with 50 mg / L Si O2 suspension for 5, 10, 20, 30, 60, 80, 100, 000, h after the detection of protein expression. The inhibitor group was incubated with HgCl2, a specific inhibitor of AQP-1 for 3 min, then stimulated with Si O2 for 2 h to detect m RNA, and stimulated for 6 h to detect protein expression. The expression of AQP-1 mRNA was detected by real-time polymerase chain reaction in each group. The expression of AQP-1 protein was detected by immunocytochemistry. [Results] Compared with the control group, the expression of AQP-1 mRNA in A549 cells treated with Si O2 for 0.5, 1, 2 and 4 h were 2.78, 3.52, 3.85 and 1.98 Fold, returned to the level of the control group at 8 h (P <0.01). The expression level of the inhibitor group was 65% of the Si02-treated group at the same stimulation time. AQP-1 protein expression: 1 Western blot results showed that compared with the control group, the expression levels of A549 cells at 3, 6, 12 and 24 h after Si O2 stimulation were 1.44, 2.56, 1.93 and 1.35 times higher than that of the control group (P <0.05 or P <0.01). The expression level in inhibitor group was lower than that in Si O2 group with the same stimulation time (70%, P <0.01). 2 Immunocytochemistry results showed that the expression of A549 cells at 3, 6, 12, 24 h after Si O2 stimulation were 1.17, 1.49, 1.29 and 1.07 times higher than that of the control group (P <0.05 or P <0.01). The expression level of inhibitor group was 71% lower than that of Si O2 group (P <0.01). [Conclusion] AQP-1 expression was increased in A549 cells induced by Si O2, and its specific inhibitor mercuric chloride inhibited the expression of AQP-1 induced by Si O2 stimulation. It is speculated that AQP-1 may be involved in the development and progression of silicosis .