大鼠肝细胞的分离及原代长期培养

目的 :研究大鼠肝细胞分离的简易方法及长期培养过程中肝细胞形态变化过程。方法 :采用体外胶原酶二步灌注法分离大鼠肝细胞 ,TB染色法计算细胞数及细胞活率。 MTT法观测新生牛血清对大鼠肝细胞增殖的影响。在含 1 0 %新生牛血清及其他附助因子的 Williams′E条件培养基中原代长期培养 ,并进行形态学的动态观察。结果 :平均每只大鼠可获取 2 .2 6× 1 0 8个肝细胞 ,平均活力为 95.6%。新生牛血清浓度与大鼠肝细胞增殖有明显的量效关系 (P<0 .0 1 )。在 Williams′E培养基中可存活 5～ 6周并保持正常形态。结论 :本方法分离的肝细胞有较高的获取率和活力 ,适合体外长期原代培养

Separation and long-term cultivation of rat hepatocytes

Objective :To study a simplified method of isolation of rat hepatocytes and to observe the pro-cess of cell morphology in long-term culture. Methods :Rat hepatocytes were isolated by a single two-stepperfusion method. The yield and viability were assessed by trypan blue exclusion. 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide] (MTT) was used to test the effect of serum concentration of newborn calf serum on the proliferation of hepatocytes. Hepatocytes were inoculated in the culture mediumconsisted of Williams' E supplemented with insulin,dexamethasone and 10％ new born calf serum. Themorphologic change of cultured hepatocytes was observed. Results:The average yield of hepatocytes was 2.26× 108 cells per rat, with an average viability of 95.6％. New born calf serum had strong biological activi-ty to stimulate the proliferation of hepatocytes and there was close-effect relationship followed by the in-crease of new born calf serum concentration. The rat hepatocytes can be cultured for 5～ 6 weeks withpreservation of normal morphologic appearance. Conclusion:The rat hepatocytes isolated by the abovemethod have high yields and viability and can be long-term cultured in vitro.