原代乳猪肝细胞低温保存的初步探索  

Hypothermic preservation of primary porcine hepatocytes

作　　者：刘鸿凌[1] 王英杰[1] 郭海涛[1] 刘国栋[1] 刘俊[1] 于乐成[1] 

机构地区：[1]重庆第三军医大学西南医院全军感染病研究所,400038

出　　处：《肝脏》2002年第3期168-170,共3页Chinese Hepatology

基　　金：全国优秀博士专项基金 (1 99947);国家自然科学专项基金资助项目 (30 0 2 70 0 1 )

摘　　要：目的　探索 4°C低温保存肝细胞的方法。方法　两步法分离新生乳猪肝细胞 ,将肝细胞分别以 3种不同低温保存液 (RPM 1 164 0培养液 ;加有聚乙二醇 (PEG)的RPM 1 164 0培养液 ;UW液 ) 4°C保存 2 4h、48h及 72h。复苏后接种培养 ,作存活率、贴壁率及细胞活性等检测 ,并观察其形态结构变化。结果　不同低温保存液保存不同时间的猪肝细胞的活力、生物学功能及形态学表现均有所不同 ,其中单用RPM 1 164 0培养液组效果最差 ,PEG组和UW组结果相似 ,均保持 70 %以上存活率达 48h ,活力与药物代谢功能无明显差别 ,镜下见复苏肝细胞浆内含丰富颗粒 ,可见较多双核细胞 ,线粒体无减少 ,粗面内质网数下降 ,滑面内质网增多。结论　猪肝细胞在 4°C可保存 48h ,加入PEG的RPM 1 164Objective To explore an optimal method for hypothermic preservation of hepatocytes at 4°C. Methods Primary porcine hepatocytes were harvested by modified two step collagenase perfusion method, then hypothermically preserved for 24h、48h or 72h before plated under culture medium conditions. The cells were stored either in the RPMI 1640 medium with or without polyethylene glycol, or in the UW(University of Wisconsin solution), the most effective solution for cold preservation of liver. After cold preservation, their viability, attachment rate and drug metabolic function were measured, and the ultrastructural changes were observed through light and electron microscopy. Results After 24h、48h or 72h of storage at 4°C in RPMI 1640 medium, cell viability and adherence rate were significant reduced, but the hepatocytes maintained in PEG or in the UW kept their function well, and their total protein secretion, extracellular LDH and drug metabolic reaction were similar to those found with fresh hepatocytes. Conclusion Primary porcine hepatocytes can be preserved for 48 hours. Addition of polyethylene glycol to the RPMI 1640 medium results in slightly greater viability and better function of hepatocytes after cold presearvation.

关 键 词：肝细胞 低温保存 聚乙二醇 UW液 人工肝 肝细胞移植 

分 类 号：R329-33[医药卫生—人体解剖和组织胚胎学]