冻存乳猪肝细胞的胶原凝胶培养研究  

Culture of cryopreservted hepatocytes of sucking pig with collagen gel

作　　者：刘鸿凌[1] 王慧芬[1] 王英杰[2] 郭海涛[2] 于乐成[2] 王宇明[2] 

机构地区：[1]解放军第302医院,北京100039 [2]第三军医大学西南医院

出　　处：《解放军医学杂志》2004年第11期967-970,共4页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学专项基金资助项目 (编号 30 0 2 70 0 1 )

摘　　要：目的　探索乳猪肝细胞的冻存以及培养条件和方法。方法　采用两步法分离乳猪肝细胞 ,液氮冻存 4个月后复苏 ,与鼠尾胶原液混合后接种培养 ,或将肝细胞接种于胶原被覆的培养瓶中 ,然后被覆上第 2层胶原培养 ,观察培养细胞的形态、白蛋白mRNA的表达、尿素合成及天冬氨酸氨基转移酶 (AST)的漏出量等。结果　冻存肝细胞的活性维持较好 ,并成功地被固定于混合凝胶或三明治形凝胶中 ,较好地保持了尿素合成以及白蛋白mRNA表达能力。结论　此冻存条件适合于猪肝细胞的长期保存 ,胶原凝胶可为肝细胞提供更接近体内的培养环境 ,较好地维持了肝细胞的功能和形态学特征。Objective To study the method of cryopreservation, and method of cultivation of hepatocytes of sucking pig. Methods Hepatocytes were isolated from sucking pig by two-step perfusion with collagenase using an extracorporeal perfusion apparatus. They were cryopreserved in liquid nitrogen for 4 months. They were thawed and seeded on gelatin coated bottom of a flask. The cells were covered with another layer of collagen. The exprssion of albumin mRNA, cellular morphology synthesis of urea, and content of oxalylacetylaminotransferase were determined in the collagen cultures. Results Thawed hepatocytes maintained their biological characters well after cryopreservation. The thawed hepatocytes could grow well in collagen gel, sandwich culture. In the 10-day cultures, typical cellular morphological and biological characteristics of cultured hepatocytes could be observed. Conclusion Hepatocytes stored in liquid nitrogen could maintain their activities for a long time. The hepatocytes could be cultivated in collagenous matrix, which provided an environment closely resembling that in nature, and their morphology and functions could be maintained.

关 键 词：肝细胞 细胞培养 低温保藏 胶原凝胶 乳猪 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]