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目的:观察小剂量TNF-α刺激下非坏死HepG2细胞是否存在HMGB1移位及释放。方法:以终浓度为25ng/ml的TNF-α作用HepG2,RAW264.7及HEK293细胞4、8、12、16、20小时和24小时,MTT法检测细胞存活率,Westernblot检测上清HMGB1含量,免疫荧光观察HMGB1移位,TUNEL法原位检测细胞凋亡。结果:TNF-α作用后24小时,HepG2,RAW264.7及HEK293细胞存活率分别为91.99%±0.30%,91.28%±0.56%和90.85%±0.72%。TNF-α作用12～24小时,HepG2和RAW264.7细胞培养上清中可以检测到明显的HMGB1,与对照组及TNF-α作用的HEK293组比较差别有统计学意义(P<0.01)。HMGB1的释放伴随着其从细胞核向胞浆的移位。TNF-α作用后24小时各组细胞凋亡率均低于5.0%,统计学无差异。结论:经TNF-α诱导,活化状态的HepG2细胞可发生HMGB1的移位及释放。
OBJECTIVE: To observe whether HMGB1 translocation and release exist in non-necrotic HepG2 cells stimulated with low dose of TNF-α. Methods: HepG2, RAW264.7 and HEK293 cells were treated with TNF-α at a final concentration of 4, 8, 12, 16, 20 hours and 24 hours respectively. The cell viability was detected by MTT assay and the content of HMGB1 in supernatant was detected by Western blot. HMGB1 translocation was observed by immunofluorescence and apoptosis was detected by TUNEL in situ. Results: The survival rates of HepG2, RAW264.7 and HEK293 cells at 24 hours after TNF-α treatment were 91.99% ± 0.30%, 91.28% ± 0.56% and 90.85% ± 0.72%, respectively. Significant HMGB1 was detectable in the culture supernatant of HepG2 and RAW264.7 cells after treated with TNF-α for 12-24 hours. The difference was statistically significant (P <0.01) between HEK293 group and control group and TNF-α group. The release of HMGB1 is accompanied by its shift from the nucleus to the cytoplasm. The apoptotic rates of all groups at 24 hours after TNF-α treatment were lower than 5.0%, with no statistical difference. Conclusion: The translocation and release of HMGB1 can occur in activated HepG2 cells induced by TNF-α.