【作者】 赵中夫； 韩德五； 刘明社； 张国英； 张芸； 杨慧；

【Author】 ZHAO Zhong-fu, HAN De-wu, LIU Ming-she, et al. Institute of hepatology Changzhi Medical College, Shanxi 046000, China; Institute of hepatology Shanxi Medical university,Shanxi 030012, China.

【机构】 长治医学院肝病研究所； 山西医科大学肝病研究所；

【摘要】 目的观察LPS诱导HMGB1在肝实质细胞与枯否细胞的表达和细胞外释放。方法培养瓶中分别培养原代肝实质细胞和枯否细胞,24 h后收获对照组和500 ng／ml LPS诱导组两种细胞,反复冻融,用半定量PT-PCR和Western blot法检测HMGB1-mRNA水平和HMGB1表达水平;接种原代肝实质细胞和枯否细胞于24孔板中,继续培养6 h、12 h、24 h和48 h,Western blot法检测各时间点对照组和 500 ng／ml LPS诱导组培养液中HMGB1含量。结果 LPS诱导24 h后,与相应对照组比较,肝实质细胞和枯否细胞HMGB1-mRNA表达水平明显增强(t=31．32和45．90,P<0．05),HMGB1蛋白表达水平也明显增强(t=46．19和38．44,P<0．05);在6 h、12 h、24 h和48 h各时间点,对照组两种细胞及诱导组肝实质细胞培养上清液中仅检测到少量HMGB1,延长培养时间,培养上清液中HMGB1含量均未见增多; 与对照组比较,诱导组枯否细胞培养液中的HMGB1含量在6 h无显著增加(t=1．48,P>0．05),但培养随时间延长,其含量明显增高(F=42．74,P<0．05),且在12 h、24 h和48 h均明显高于对照组(t分别= 21．95,32.39,44．16,P<0．05)。结论 LPS可诱导肝实质细胞和枯否细胞HMGB1表达增强,肝实质细胞不主动释放HMGB1,而枯否细胞能主动释放HMGB1到细胞外。更多还原

【Abstract】 Objective To investigate HMGB-1 expression and extracellular release of primary cultural hepatic parenchymal cells and Kupffer’s cells that were induced by LPS. Methods Primary hepatic parenchymal cells and Kupffer’s cells were cultured in culture flasks, and the cells which were treated by 500 ng/ml LPS for 24 hours and which not induced were harvested as the induced groups and the control groups. All of cells were repeatedly freeze-thawed, and the expression levels of HMGB1-mRNA and HMGB1 protein were detected by semi-quantitative RT-PCR and Western blot respectively. Then subcultured hepatic parenchymal cells and Kupffer’s cells into 24-well Culture plates for 6 h, 12 h, 24 h and 48 h, and the HMGB1 protein in culture fluids were detected by Western blotat each time point. Results Compared with control groups, the expression levels of HMGB1-mRNA in the induced groups were significantly increased in both HC and KC at 24 h(t = 31.32 and 45.90, P < 0.05) and the protein levels of HMGB1 showed the same results(t = 46.19 and 38.44, P < 0.05). There were a small quantity of HMGB1 protein in the two control groups and the induced group of hepatic parenchymal cells culture fluids. However the HMGB1 protein in induced group of Kupffer’s cells were obviously increased with the culture time lasting(F = 142.74, P < 0.05). Compared the with control group, the level of HMGB1 protein in induced group of Kupffer’s cells was not raised at 6 h (t = 9.57, P > 0.05) but were significantly increased at 12 h, 24 h and 48 h respectively(t = 21.95, 32.39, 44.16, P < 0.05). Conclusion LPS could increase HMGB1 expressing in HC and KC and HMGB1 releasing from KC, but not from HC. The results suggest that KC play an important role in triggering inflammation and liver injury.更多还原