大鼠原代培养肝细胞的生物学特性研究

BIOLOGICAL STUDIES OF PRIMARY CULTURED RAT HEPATOCYTES

目的 :观察培养的原代大鼠肝细胞形态学和功能的动态变化 ,探讨其短期培养内的最佳功能状态。方法 :采用Seglen胶原酶二步灌流法分离肝细胞。光镜、透射电镜和免疫组化方法观察细胞形态学改变 ;并收集不同时期培养上清监测生化指标的改变。结果 :平均每只大鼠肝获取 1.2 1× 10 8个肝细胞 ,平均活力为 93.6 %。光镜下见肝细胞生长良好 ,可存活 3周 ,电镜下可见丰富的细胞器 ,细胞连接及胆小管 ,细胞角蛋白 18强阳性表达。肝细胞功能检测白蛋白分泌及尿素合成功能在第 3,4天时较好。结论 :肝细胞的形态和功能具有一致性 ,原代肝细胞在培养第 3,4天功能较好 ,可能为肝细胞移植和生物人工肝应用的最佳时机。

Objective: To observe the process of alteration of cell morphology and function in cultured primary rat hepatocytes and to investigate the optimal functional status of them in one week. Methods: Rat hepatocytes were isolated by the modified two-step method described by Seglen. The morphologic change of cultured hepatocytes was observed by transmission electron microscope and immunohistochemistry; the concentrations of albumin and urea in the supernatant in different cultural period were examined. Results:The average yield of rat hepatocytes was 1.21 ×10 8 cells per rat with an average viability of 93.6%. Hepatocytes grew well and survived for three weeds. Under electron microscope, the abundance of organelles and particular ultrastructure of hepatocytes were found. Our data showed albumit excretion and urea synthesis level had fluctuating change in one week, and the higher albumin synthesis and urea level were found at the third and fourth day.Conclusion: These data indicate that primary cultured rat hepatocytes function well in the third and fourth day,which may be the optimal time of hepatocytes for liver cell transplantation and bioartificial liver support system.