长期培养的大鼠原代肝细胞功能和形态学观察

Investigation on the function and morphometrics of cultured primary rat hepatocytes

目的研究大鼠原代肝细胞长期体外培养后功能和形态的变化。方法采用两步胶原酶原位灌流法分离大鼠肝细胞,并用Percoll分离液进行密度梯度离心进一步纯化肝细胞,采用0.4%台盼蓝染色观察细胞活力。然后将细胞接种于HepatoZYME-SFM培养基中培养,定期收集肝细胞培养液上清检测丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、白蛋白、尿素氮的水平。采用乙氧基试卤灵O-脱乙基酶活性(EROD)方法检测肝细胞P450的CYPⅠA1功能。结果新鲜分离的大鼠肝细胞总数(2-3)×108cells/wholeliver,Percoll分离液纯化后活力和纯度在90%以上。HepatoZYME-SFM培养下肝细胞生长良好并保持正常形态。AST、ALT水平在培养3d后下降显著,6-9d后趋于相对稳定的低水平。白蛋白的分泌功能、尿素合成能力在18d内维持在较高水平。可在3-6d检测到CYPⅠA1酶活性。结论Percoll液纯化新分离肝细胞可提高其活率和纯度,HepatoZYME-SFM培养条件下肝细胞可有效保持良好形态结构和一定的生物合成代谢能力,适合肝细胞的体外长期培养和功能研究。

AIM: To investigate the function and morphological changes of long-term cultured primary rat hepatocytes. METHODS: Rat primary hepatocytes were isolated by two-step in situ collagenase perfusion method, and then were purified by density and grade centrifugal method with Percoll. Cell viability was observed by 0.4% trypan blue. The hepatocytes were seeded into 6 wells plate with HepatoZYME-SFM medium. ALT, AST, albumin and urea levels in the supernatant were measured, CYPⅠA1 was detected with EROD method. RESULTS: (2-3)×108 cells per whole liver were obtained with viability and purity above 90% after purified with Percoll. Hepatocytes cultured in HepatoZYME-SFM grew well with normal hepatocyte morphometrics. ALT, AST levels in the supernatant decreased after 3-day culture, and kept at a stable level after 6-9 days. Albumin secretion and urea synthesis were maintained at high levels in 18 days, while CYPⅠA 1 enzyme activity was only detected in 3-6 days. CONCLUSIONS: Percoll was used to increase the viability and purity of freshly isolated rat hepatocytes. Hepatocyte morphometrics and their biological synthesized function are effectively maintained in HepatoZYME-SFM medium.