大鼠原代肝细胞的形态和功能

Morphology and function of primarily cultured rat hepatocytes

目的 :对分离培养的原代大鼠肝细胞进行形态学观察和功能研究 ,探讨其短期培养内的最佳功能状态 ,以便更好地用于肝细胞移植或生物人工肝 .方法 :采用Seglen胶原酶二步灌流法灌注SD雄性大鼠肝脏 ,获取的肝细胞在含有多种辅助因子的Williams’E培养基中进行培养 .台盼蓝排斥法计算细胞产量和细胞活率 ;光镜下直接或HE染色后动态观察细胞形态学改变 ,透射电镜观察其超微结构 ;并收集不同时期培养上清检测其细胞分泌等功能 .结果 :每只大鼠肝平均可获取 1 .2 1× 1 0 8个肝细胞 ,平均活力为 93.6 % .光镜下可见肝细胞呈多角形生长 ,有双核 ,连接成片状或岛状 .电镜下可见内质网、车轮状线粒体、糖原颗粒、细胞连接及胆小管 .肝细胞功能检测显示LDH漏出量、白蛋白分泌及尿素合成功能在 1wk内出现波动性变化 ,第 3日LDH漏出量最低 ,白蛋白分泌及尿素合成功能较好 .结论 :离体培养的 3d原代肝细胞具有较好的功能 。

AIM: To observe the process of the alteration of cell morphology and function in primarily cultured rat hepatocytes and to investigate their optimal functional condition for hepatocyte transplantation and bioartificial liver support system. METHODS: Rat hepatocytes were isolated by the modified two step method described by Seglen. The yield and viability were assessed by trypan blue exclusion. Hepatocytes were inoculated in the culture medium consisting of Williams’E supplemented with insulin,glycogen, transferrin,dexamethasone,nicotinamide and 10 g·L -1 fetal bovine serum. The morphologic changes of cultured hepatocytes were observed; the ultrastructural study was conducted and the concentrations of albumin, urea and LDH (lactate dehydrogenase) 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 were observed under light microscope, polygon hepatocytes,some of which were binuclear, were aggregated and arranged in groups or cords. Under electron microscope, abundance of organelles and particular ultrastructure of hepatocytes were found. Our data showed that LDH leakage, albumin synthesis and urea level had fluctuating changes in one week,but the third day found less LDH leakage and higher albumin synthesis and urea level. CONCLUSION: Primarily cultured rat hepatocytes function well on the third day, which may be the optimal time for transplantation and bioartificial liver support system.