肝细胞凋亡模型的建立

目的　建立体内、外肝细胞凋亡模型 ,为进一步研究药物对肝细胞凋亡的调控作用奠定基础。方法　 (1)采用D GalN +LPS小鼠腹腔注射建立体内肝细胞损伤模型 ;(2 )采用H2 O2 (0 0 5～ 0 4mmol·L-1)与原代大鼠肝细胞共培养建立体外肝细胞损伤模型 ,分别用形态学、DNA电泳和流式细胞术检测等方法。结果　 (1)D GalN(70 0mg·kg-1,ip) +LPS(1μg·kg-1,ip)可明显升高小鼠血中TNF水平和MDA含量 ,使肝线粒体Mn SOD活性降低 ,小鼠肝细胞皱缩变小、核染色质凝聚和DNA片段化 (DNA梯状条带 ) ;(2 )H2 O2(0 1mmol·L-1,1h)可致大鼠肝细胞增殖受抑、肝细胞MDA含量升高 ,肝细胞DNA的AO荧光染色变亮 ,DNA电泳呈片段化 ,流式细胞术肝细胞DNA亚G1峰 (即凋亡峰 )明显升高。结论　采用D GalN +LPS与低浓度H2 O2 可分别建立较理想的体内、外肝细胞凋亡模型

Establishment of the models of hepatocyte apoptosis in vivo and in vitro

AIM To establish apoptosis models of hepatocytes in vivo and in vitro ,for further studying regulative effects of drugs on apoptosis hepatocytes METHODS (1) A model of hepatocyte apoptosis in vivo was established by injecting D GalN(700 mg·kg -1 )+LPS(1 μg·kg -1 ) intraperitoneally (2) Another model of hepatocyte apoptosis in vitro was established by primary hepatocytes coincubated with H 2O 2(0 05～0 4 mmol·L -1 ) In addition to the observation of the morphological changes, electrophoresis of extrated DNA and flow cytometry were used RESULTS (1)The elevation of TNF level and MDA content, the loss of Mn SOD activity, murine hepatocyte shrinkage, chromatin condensation and DNA fragmentation(DNA ladder) were induced at the end of 5 h after injection of D GalN+LPS in mice (2)The suppression of proliferation, the elevation of MDA content of hepatocytes from rats, chromatin condensation, DNA fragmentation and the increased apoptosis peaks were induced by H 2O 2(0 1 mmol·L -1 ,1 h) CONCLUSION The ideal models of hepatocytes apoptosis in vivo or in vitro may be separately established by D GalN+LPS or H 2O 2