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目的:观察缺氧对喉癌细胞高迁移率族蛋白B1(HMGB1)释放的诱导作用,并探讨其可能的机制。方法:采用人喉癌细胞株Hep-2,观察缺氧(1%O2)培养不同时间对细胞培养上清液中HMGB1含量、细胞HMGB1蛋白和mRNA的影响,及不同浓度的MAPK信号通路抑制剂(PD98059、SP600125、SB202190)、NF-κB抑制剂(PDTC)对缺氧诱导的HMGB1释放的影响。上清液HMGB1含量和细胞HMGB1蛋白表达水平分别采用酶联免疫吸附试验和Western blot检测;细胞HMGB1mRNA表达水平采用实时荧光定量PCR法检测。结果:培养上清液中HMGB1含量和细胞HMGB1蛋白表达水平均于缺氧诱导12h后升高,呈时间依赖性;细胞HMGB1mRNA表达水平于缺氧诱导6h开始升高,并随诱导时间延长而进一步升高;20μmol/L PD98059、SP600125和50mg/L PDTC对缺氧诱导的HMGB1释放有不完全的抑制作用。结论:缺氧诱导喉癌细胞释放HMGB1,其机制可能与MAPK/NF-κB信号通路有关。
Objective: To observe the induction of hypoxia on the release of high mobility group box 1 (HMGB1) in laryngeal carcinoma cells and to explore its possible mechanism. Methods: Human laryngeal carcinoma cell line Hep-2 was used to observe the effects of hypoxia (1% O2) on the content of HMGB1 and HMGB1 protein and mRNA in cell culture supernatant and the effect of different concentrations of MAPK signal pathway inhibitor (PD98059, SP600125, SB202190) and NF-κB inhibitor (PDTC) on hypoxia-induced HMGB1 release. The content of HMGB1 in the supernatant and the level of HMGB1 protein in the cells were detected by ELISA and Western blot, respectively. The expression of HMGB1 mRNA was detected by real-time fluorescence quantitative PCR. Results: The content of HMGB1 and the level of HMGB1 protein in culture supernatants were increased 12h after hypoxia induction in a time-dependent manner. The expression level of HMGB1 mRNA in HUVECs increased from 6h after hypoxia induction and further increased with the induction time 20μmol / L PD98059, SP600125 and 50mg / L PDTC had an incomplete inhibitory effect on hypoxia-induced HMGB1 release. Conclusion: Hypoxia induces the release of HMGB1 in laryngeal carcinoma cells, which may be related to the MAPK / NF-κB signaling pathway.