Zhao Zhong-fu, Han De-wu, Liu Ming-she, Zhang Guo-ying, Zhang Yun, Yang Hui, Yang Liu-xu
Institute of Hepatology, Changzhi Medical College, Shanxi 046000, China.
Zhonghua Gan Zang Bing Za Zhi. 2007 Sep;15(9):676-80.
To investigate HMGB-1 expression and its extracellular release of cultured primary hepatic parenchymal cells (HC) and Kupffer cells (KC) that were induced by lipopolysaccharides (LPS).
Primary hepatic parenchymal cells and Kupffer cells were cultured in flasks, and some cells were treated with 500 microg/L LPS for 24 hours (induced group) and some were not treated with LPS and served as controls. All of the cells were repeatedly frozen-thawed, and the expression levels of HMGB1-mRNA and HMGB1 proteins were detected by semi-quantitative RT-PCR and Western blot respectively. Then HC and KC were subcultured in 24-well culture plates for 6 h, 12 h, 24 h and 48 h, and the HMGB1 protein in culture fluids was detected by Western blot at each time point.
Compared with the cells in the control group, the expression levels of HMGB1-mRNA in the induced group 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 was a small quantity of HMGB1 protein in the culture fluids of two control groups and the induced group of HC. However the HMGB1 protein in the induced group of KC were obviously increased with prolonged culture time (F=42.74, P<0.05). Compared with the control group, the level of HMGB1 protein in the induced group of KC was not increased at 6 h (t=9.57, P>0.05) but was significantly increased at 12 h, 24 h and 48 h (t=21.95, 32.39, 44.16, respectively P<0.05).
LPS could increase HMGB1 expression of HC and KC and HMGB1 release from KC, but not from HC. The results suggest that KC play an important role in triggering inflammation and liver injury.
研究脂多糖（LPS）诱导培养的原代肝实质细胞（HC）和库普弗细胞（KC）中高迁移率族蛋白B1（HMGB-1）的表达及其细胞外释放情况。
将原代肝实质细胞和库普弗细胞培养于培养瓶中，部分细胞用500μg/L LPS处理24小时（诱导组），部分细胞未用LPS处理作为对照。所有细胞反复冻融，分别采用半定量逆转录聚合酶链反应（RT-PCR）和蛋白质印迹法检测HMGB1-mRNA和HMGB1蛋白的表达水平。然后将HC和KC接种于24孔培养板中培养6小时、12小时、24小时和48小时，在每个时间点采用蛋白质印迹法检测培养液中的HMGB1蛋白。
与对照组细胞相比，诱导组HC和KC在24小时时HMGB1-mRNA表达水平均显著升高（t分别为31.32和45.90，P<0.05），HMGB1蛋白水平也呈现相同结果（t分别为46.19和38.44，P<0.05）。HC对照组和诱导组培养液中均有少量HMGB1蛋白。然而，KC诱导组的HMGB1蛋白随培养时间延长明显增加（F=42.74，P<0.05）。与对照组相比，KC诱导组在6小时时HMGB1蛋白水平未升高（t=9.57，P>0.05），但在12小时、24小时和48小时时显著升高（t分别为21.95、32.39、44.16，P均<0.05）。
LPS可增加HC和KC中HMGB1的表达以及KC中HMGB1的释放，但不能增加HC中HMGB1的释放。结果提示KC在引发炎症和肝损伤中起重要作用。
