肿瘤坏死因子α调节人肝癌MHCC-97H细胞中白细胞介素8的分泌

TNFα induced IL-8 production through p38 MAPK- NF-r.B pathway in human hepatoceilular carcinoma cells

目的研究肿瘤坏死因子（TNF）仅诱导入肝癌细胞系MHCC-97H分泌白细胞介素8（IL-8）及p38丝裂原活化蛋白激酶（MAPK）和核因子-KB（NF—KB）信号通路对其的调节作用。方法酶联免疫吸附法检测不同浓度的TNFα（0、1、5ng／ml）干预人肝癌细胞MHCC-97H24h和相同浓度的TNFα（1ng／m1）干预不同时间后，细胞上清液中IL-8的浓度；Westemblot和免疫荧光方法检测TNFα（1ng／m1）刺激MHCC-97H细胞后，p38MAPK磷酸化蛋白的表达及其分布；非放射性NF-κBp50／p65转录因子潘胜试剂盒检测TNFα干预MHCC-97H细胞后核蛋白中活性NF—κBp65蛋白的含量。多组均数比较采用单因素方差分析，多个样本均数间两两比较用q检验。结果TNFα明显促进肝癌细胞分泌IL-8，具有显著的时间和浓度依赖性：TNFα干预0、6、12、18、24h，IL-8的浓度分别为（47．45±9．78）pg／ml、（497．41±52．83）pg／ml、（694．14±44．43）pg／ml、（909．35土97．22）pg／ml、（1093．59±75．72）pg／ml（F=144．04，P〈0．01）；TNFα0、1、5、10ng／ml干预24h，IL-8浓度分别为（47．45±9．12）pg／ml、（1093．59±75．72）pg／ml、（1372．77±85．10）pg／ml和（1614．55±153．52）pg／ml（F=364．14，P〈0．01）。TNF仅刺激肝癌细胞后，p38MAPK磷酸化蛋白表达明显上调，给予p38MAPK通路特异性抑制剂（SB203580）可以显著抑制TNFα诱导的IL-8的分泌（P值均〈0．01）且呈浓度依赖性（F=165．92，P〈0．01）。免疫荧光结果显示TNFⅨ促进肝癌细胞NF—κBp65的核转位，且呈浓度依赖性（TNFα0、1、5、10ng／ml处理肝癌细胞1h后细胞核蛋白表达NF—KBp65对应吸光度值分别为0．52±0．04、2．75±0．15、3．13±0．18、3．81±0．14（F=1266．42，P〈0．05），而SB203580则可以部分抑制NF-KBp65的核转位（F=141．20，P〈0．05）。结论TNFα通过p38MAPK—NF—κB信号途径调节人肝癌细胞系MHCC-97分泌IL-8。

Objective To identify the role of p38 MAPK- NF-κB signaling pathway in TNF-αinduced IL-8 production in human hepatocellular carcinoma cells. Methods The concentrations of IL-8 from MHCC- 97H cells were measured by an enzyme-linked immunosorbent assay （ELISA）. The phosphorylation of p38 MAPK was analyzed by Western blot and immunofluorescence. NF-κB p65 protein nuclear translocation was determined by non-radioactive NF-κB p50 / p65 transcription factor activity kit and immunofluorescence. Results The IL-8 production from MHCC-97H cells challenged with TNFa significantly increased in a time- dependent （ F = 144.04, P 〈 0.01） and dose-dependent （ F = 364.14, P 〈 0.01） manners, as compared withthose without TNFct challenge. TNFα up-regulated the phosphorylation levels of p38 MAPK and increased the translocation of NF-κB p65 protein into the nucleus, also proved by immunofluorescence staining, p38 MAPK inhibitor （SB203580） could significantly inhibit IL-8 production in a dose-dependent manners （ F = 65.47, P 〈 0.01）, and partially inhibited NF-κB p65 nuclear translocation in a dose-dependent manner （F = 141.20, P 〈 0.05）. Conclusion TNF-α could increase the production of IL-8 in MHCC-97H cells and p38 MAPK- NF-κB pathways seem to play a central role in the regulation of IL-8 production.