维持静息细胞周期状态的小鼠肝细胞的分离纯化和原代培养

Isolation,purification and primary culture of mouse hepatocytes in quiescent cell cycle state

目的建立维持正常肝细胞静息细胞周期状态的小鼠肝细胞的分离纯化及原代培养方法。方法对传统小鼠肝细胞分离纯化的原位两步胶原酶灌流法进行改良,台盼蓝染色法计数细胞总数并鉴定肝细胞活率;用改良的WME培养基对C57BL/6小鼠肝细胞进行原代培养,经过碘酸雪夫氏染色(Periodic acid-Schiff stain,PAS)和全自动生化分析仪鉴定肝细胞的生物学特性及功能;Western blot法检测分离纯化后的小鼠肝细胞及原代培养24、48、72、96 h小鼠肝细胞的细胞周期蛋白p16、cyclin D1和cyclin A的表达水平。结果分离纯化后可从每只小鼠肝脏稳定获取(3～5)×107个肝细胞,细胞活率达(86.68±2.46)%;原代培养96 h内的小鼠肝细胞能保持合成糖原、白蛋白、尿素的功能;原代培养小鼠肝细胞中,细胞周期蛋白p16、cyclin D1和cyclin A的表达水平与分离纯化的小鼠肝细胞相比,差异无统计学意义(P>0.05),表明原代培养96 h内的小鼠肝细胞均能维持静息细胞周期状态。结论改良的分离纯化方法能稳定获得高产量、高活率的小鼠肝细胞;改良后的原代培养方法可使肝细胞维持其特异性功能、分化特性及静息细胞周期状态。

Objective To develop the methods for isolation and primary culture of mouse hepatocytes in quiescent cell cycle state.Methods Conventional two-step collagenase perfusion technique in situ for isolation and purification of mouse hepatocytes was improved.Trypan blue staining was used for total cell counting and determination of survival rate of hepatocytes.The hepatocytes of C57BL / 6 mice were subjected to primary culture in modified WME medium and analyzed for biological characters and function by periodic acid-Schiff（PAS） stain and full-automatic biochemical analyzer.The expression levels of cell cycle proteins such as p16,cyclin D1 and cyclin A in purified mouse hepatocytes and in the hepatocytes 24,48,72 and 96 h after primary culture were determined by Western blot.Results About（3 ～ 5） × 107 hepatocytes were obtained from each mouse,of which the survival rate was（86.68 ± 2.46）%.The mouse hepatocytes within 96 h after primary culture maintain the functions of synthesis of glycogen,albumin and urea,in which the expression levels of p16,cyclin D1 and cyclin A showed no significant difference with those in purified mouse hepatocytes（P 0.05）,indicating a quiescent cell cycle state.Conclusion Mouse hepatocytes with high output and survival rate were obtained by the improved method for isolation and purification,and the hepatocytes in primary culture by improved method maintain their specific function,differential property and quiescent cell cycle state.