大鼠骨髓间充质干细胞体外诱导分化为成熟肝细胞样细胞  

Differentiation of rat bone marrow mesenchymal stem cell into mature hepatocyte-like cell in vitro

作　　者：舒赛男[1] 魏来[2] 展玉涛[2] 方峰[1] 王宇[2] 

机构地区：[1]华中科技大学同济医学院同济医院儿科,湖北武汉430030 [2]北京大学人民医院肝病研究所,北京100044

出　　处：《基础医学与临床》2006年第9期999-1003,共5页Basic and Clinical Medicine

基　　金：国家高技术研究发展计划项目(2001AA216031);中国博士后科学基金(2005037676);湖北省科技攻关计划项目(2005AA301C14)

摘　　要：目的 建立和优化大鼠骨髓间充质干细胞（MSCs）分离培养方法 ,探讨其肝系分化条件及潜能.方法分离5～6周龄大鼠骨髓细胞,Percoll密度梯度离心得到单个核细胞,贴壁法培养和纯化MSCs.用形态学观察、反转录聚合酶链反应和免疫细胞化学法探讨培养底物、细胞因子种类与浓度对MSCs肝分化潜能的影响.结果 57%Percoll密度梯度离心能在骨髓单个核细胞分离的质和量上达到最佳.贴壁24h后去悬浮细胞、低接种密度传代培养可获增殖能力强、功能活跃的MSCs.MSCs在10 mg/L纤维连接蛋白为底物、HGF/FGF-4诱导下,纤维状细胞渐变为圆形,表达白蛋白及其mRNA,不表达甲胎球蛋白.结论 优化大鼠周龄、分离液的密度、细胞培养方法有助于获得多向分化潜能保持良好的MSCs.MSCs在HGF/FGF-4诱导下呈成熟肝细胞样细胞表现.Objective To establish and optimize the method of isolation and cultivation of rat bone marrow mesenchymal stem cells （MSCs） , and to investigate the condition and capability of hepatic differentiation of MSCs. Methods Rat bone marrow cells were collected from rats of 5-6 week and mononeuclear cells were isolated by Percoll density gradient centrifugation. MSCs were cultivated and purified by adherence method. Morphology, RTPCR and immunocytochemistry were used to identify the role of substrates, the kinds of the cytokines and concentrations of cytokines on the hepatic differentiation potential. Results The quality and quantity of the isolated bone marrow mononuclear cells reached the peak by using 57% Percoll density gradient centrifugation. Discarding the suspending cells after 24h incubation and subculturing the cells with low seeding density were helpful for acquiring MSCs with improved reproductive capability and active function. MSCs could be successfully induced under HGF/ FGF-4 induction on the substrate of 10 μg/mL fibronectin. MSCs exhibited round in shape after differentiation, instead of fibroblast-like morphology before. Albumin mRNA and protein were positively expressed in MSCs, without detection of alpha-fetoprotein （AFP）. Conclusion The optimization of rats＇ age, the density of Percoll, the methods of cell cultivation are conducing to obtaining a pure population of MSCs with good differential activity. MSCs are inclined to differentiate into mature hepatocyte -like cells under the induction of HGF/FGF-4.

关 键 词：间充质干细胞 骨髓 肝分化潜能 

分 类 号：Q813[生物学—生物工程]