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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
OBJECTIVE: To study a simple method of rat hepatocyte isolation and morphological changes of hepatocytes during long-term culture. Methods: Rat liver cells were isolated by collagenase two - step perfusion method. The number of cells and cell viability were calculated by TB staining. Effect of Newborn Bovine Serum on Proliferation of Rat Hepatocytes by MTT Method. Primary culture was performed in Williams’E conditioned media containing 10% newborn calf serum and other accessory factors for dynamic, morphological observation. RESULTS: On average, 2.26 × 108 hepatocytes were obtained per rat, with an average viability of 95.6%. Newborn calf serum concentration and rat liver cell proliferation has a significant dose-effect relationship (P <0. In Williams’E medium can survive 5 to 6 weeks and maintain normal morphology. Conclusion: The hepatocytes isolated by this method have higher acquisition rate and viability and are suitable for long-term primary culture in vitro