骨髓间充质干细胞维持猪肝细胞形态与功能的实验研究  

Experimental study on the maintenance of porcine hepatocyte morphology and function by culturing with bone marrow mesenchymal stem cells

作　　者：顾劲扬[1] 施晓雷[2] 张悦[1] 丁义涛[2] 

机构地区：[1]南京医科大学鼓楼临床医学院肝胆外科,210008 [2]南京大学医学院附属鼓楼医院肝胆外科 ,210008

出　　处：《中华肝胆外科杂志》2010年第2期130-133,共4页Chinese Journal of Hepatobiliary Surgery

基　　金：基金项目：国家自然科学基金（30772129）

摘　　要：目的建立猪肝细胞与骨髓间充质干细胞（MSCs）体外共培养体系，为生物人工肝的构建提供理想细胞来源。方法自中华实验猪（n=3）髂前上棘抽取骨髓，采用密度梯度离心法分离单个核细胞，贴壁传代培养至第3代；原位两步胶原酶法分离猪肝细胞后与MSCs随机混合培养，观察共培养肝细胞形态和功能的变化水平。结果第3代MSCs纯度〉90％，肝细胞活率〉95％，纯度〉99％。共培养组肝细胞迅速黏附于MSCs表面，在三维空间呈球形聚集生长，异质细胞间出现细胞连接，超微结构与正常肝细胞接近。共培养组肝细胞白蛋白分泌水平和尿素合成能力自第1天培养起均显著高于对照组（P〈O．05），并在第2天达到高峰。结论猪肝细胞与MSCs共培养可维持肝细胞形态与功能，使构建功能性生物人工肝成为可能。Objective To establish porcine hepatocyte co-culture system with bone marrow mes- enchymal stem cells （MSCs） in vitro for the ideal cell source of bioartificial liver. Methods Mononu- clear cells were isolated from bone marrow aspirate extracted from the anterior superior iliac spine of swines （n=3） by 1. 077 g/ml density gradient eentrifugation. MSCs of passage 3 and primary hepato- cytes harvested by a two-step in situ collagenase perfusion teehnique were randomly distributed, and the morphological and functional changes of heterotypic interactions were charaeterized. Results The purity of the third passage MSCs and primary hepatoeytes was more than 90~ and 99 ~, respectively. Hepatocyte viability was greater than 95 %. A rapid attachment and self-organization of three-dimen- sional hepatocyte spheroids were encouraged in co-culture. Heterotypic junctions remained similar to that of hepatocytes in vivo. Hepatocyte performance levels such as albumin secretion and urea synthe- sis were all significantly enhanced in co-culture group compared with hepatocyte homo-culture （P（0.05）. The best hepatic function levels were achieved on day 2 and moderately decreased in the following co-culture days. Conclusion Co-cultivation of porcine hepatocytes and MSCs may preserve hepatocyte morphology and function, which could contribute to the functional bioartificial liver.

关 键 词：肝功能衰竭 猪肝细胞 骨髓间充质干细胞 共培养 形态 功能 

分 类 号：R318.14[医药卫生—生物医学工程]