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目的探讨细胞因子白介素-1β(IL-1β)对人肺腺癌细胞株A549细胞环氧合酶2(COX-2)表达的影响及调节机制。方法A549细胞培养达80%融合后,用不同浓度的IL-1β(0、0.1、1、5和10ng/mL)干预或用5ng/mLIL-1β干预不同时间(0、3、6、9、12和24h)观察白介素-1β(IL-1β)对A549细胞COX-2表达的影响;用5ng/mLIL-1β与有丝分裂原激活的细胞外调节激酶(ERK)抑制剂PD098059、P38有丝分裂原激活的蛋白激酶(MAPK)抑制剂SB203580、蛋白激酶C(PKC)抑制剂H-7共同干预A549细胞观察IL-1β诱导A549细胞COX-2表达的信号传导途径;用COX-2系列报告基因质粒及对照质粒转染A549细胞后予以IL-1β干预,以不加IL-1β干预的细胞为对照组观察IL-1β对COX-2启动子的影响;Westernblot检测COX-2蛋白表达,RT-PCR检测COX-2mRNA表达。COX-2报告基因活性应用荧光素酶活性分析法测定。结果A549细胞COX-2蛋白表达和mRNA表达呈IL-1β浓度依赖性表达增加,干预9h表达达高峰;SB203580、H-7可明显抑制IL-1β诱导A549细胞COX-2表达,PD098059对IL-1β诱导A549细胞COX-2表达无影响;IL-1β对COX-2报告基因无影响。结论PKC途径和P38MAPK途径参与IL-1β诱导A549细胞COX-2蛋白表达。
Objective To investigate the effect of interleukin-1β (IL-1β) on the expression of cyclooxygenase 2 (COX-2) in human lung adenocarcinoma cell line A549 and its regulatory mechanism. Methods A549 cells were cultured with 80% confluency and treated with different concentrations of IL-1β (0, 0.1, 1, 5 and 10 ng / mL) or with 5 ng / mL IL-1β for different times The effect of interleukin-1β (IL-1β) on the expression of COX-2 in A549 cells was observed by flow cytometry (FCM), 12 and 24 hours respectively. The effect of IL-1β on mitogen-activated protein kinase IL-1βinduced the expression of COX-2 in A549 cells by interfering with A549 cells via SB203580, a protein kinase (MAPK) inhibitor H-7, and protein kinase C (PKC) inhibitor H-7. COX-2 reporter plasmids and control The effect of IL-1β on the COX-2 promoter was observed by Western Blot. The expression of COX-2 was detected by RT-PCR -2 mRNA expression. The COX-2 reporter gene activity was measured by luciferase activity assay. Results The expression of COX-2 protein and mRNA in A549 cells increased in a dose-dependent manner and reached the peak at 9 hours after intervention. SB203580 and H-7 significantly inhibited the COX-2 expression in A549 cells. The effect of PD098059 on IL- 1β induced COX-2 expression in A549 cells had no effect; IL-1β had no effect on COX-2 reporter gene. Conclusion The PKC pathway and P38MAPK pathway are involved in the COX-2 protein expression in A549 cells induced by IL-1β.