摘要
背景:在成年动物肝脏中干细胞含量很少,且体外大量扩增并保持未分化状态的问题仍然没有很好的解决,许多科研者预测胚胎肝干细胞可能具有诱人的临床应用价值。近年来C-kit被选择作为研究肝干细胞的共同标志,并发现C-kit抗原参与肝干细胞的生长和发育。目的:对胎儿肝脏来源的C-kit+细胞进行分离培养,观察其体外生长和分化的特征。设计、时间及地点:细胞学体外观察,于2005-10/2008-03在南京大学医学院和国家生物医药技术重点实验室完成。材料:肝脏C-kit+细胞来源于32例流产胎儿,由南京大学医学院附属鼓楼医院提供。Percoll分离液为Pharmacia公司产品,Ficoll分离液为天津灏阳生物制品科技有限责任公司产品,表皮生长因子、肝细胞生长因子为R&D公司产品。方法:无菌状态下取出胎儿肝脏,以胶原酶消化法和机械分离法获得胎儿肝脏来源单细胞悬液,分别采用1.070g/mLPercoll分离液和1.077g/mLFicoll分离液进行密度梯度离心,吸取界层细胞,添加含体积分数为0.1FBS、20μg/L肝细胞生长因子、40μg/L表皮生长因子的DMEM/F12新鲜培养基诱导9d。主要观察指标:胎儿肝脏中C-kit+细胞计数,界层细胞形态、生长特征、表型及C-kit标志的鉴定,界层细胞诱导分化结果。结果:未经分离的新鲜胎儿肝脏细胞中C-kit+细胞所占比例仅为1.28%,使用Percoll和Ficoll分离液获取的界层细胞分布在三角形区域内的C-kit+细胞所占比例显著增加,分别为74.21%和72.15%,但绝对数量仍然有限。两种分离液获取的界层细胞在形态、生长状况方面基本相似,免疫细胞化学染色鉴定后大体可分为4类:第1类细胞为C-kit+细胞集落,第2类细胞既呈CD29+、CD90+、CD34-、C-kit-(间充质干细胞表型特征),又较弱地表达细胞角蛋白19(胆管细胞特异性标志),第3类细胞较强地表达细胞角蛋白19,不表达C-kit抗原,第4类细胞高表达CD45,微弱表达CD34、CD90、C-kit。诱导后,界层细胞角蛋白19呈强阳性表达,不表达C-kit、白蛋白、甲胎蛋白及细胞角蛋白18。结论:胎儿肝脏中的C-kit+细胞数量很少,Percoll和Ficoll分离液均可提高C-kit+细胞的得率。胎儿肝脏中可能存在的一类间充质-胆管细胞的中间态细胞,这种细胞可能有促进C-kit+细胞生长的作用。间充质干细胞可向胆管细胞分化。
BACKGROUND： Stem cells are few in adult animal liver, and problems on in vitro amplification and undifferentiation still do not solved. Many scholars have predicted that embryonic hepatic stem cells have inducible clinical application. Presently, C-kit has been selected as a common marker of studing hepatic stem cells. It is found that C-kit antigen participates in growth and development of hepatic stem cells. OBJECTIVE： To investigate the methods of isolation and culture condition of C-kit＋ cells in human fetal liver and the characteristics of growth and differentiation in vitro. DESIGN, TIME AND SETTING： The cytology in vitro observation was performed at the Medical School ＆ State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University from October 2005 to March 2008. MATERIALS： Liver C-kit＋ cells were obtained from 32 aborted fetal samples at the Drum Tower Hospital of Medical School of Nanjing University. Percoll separating medium was supplied by Pharmacia Company. Ficoll separating medium was purchased from Haoyang, China. Epidermal growth factor and hepatocyte growth factor were bought from R＆D Company. METHODS： Fetal liver was obtained sterilely. Monoplast suspension was collected by collagenase digestion and mechanical separation, and then centrifuged using 1.070 g/mL Percoll separating medium and 1.077 g/mL Ficoll separating medium. Cells achieved from the interface of separatory liquids were cultured in DMEM/F12 medium, supplemented with 0.1 volume fraction of fetal bovine serum, 20 u g/L hepatocyte growth factor and 40 u g/L epidermal growth factor, for 9 days. MAIN OUTCOME MEASURES： Number of C-kit＋ cells, characteristics, morphology, growth and differentiation of cells achieved from the interface, phenotype and C-kit markers of cells. RESULTS： Proportion of C-kit＋ cells was 1.28% in non-separation hepatocytes. Ficoll and Percoll could improve the harvest of C-kit＋ cells derived from fetal liver, and the actual number of C-kit＋ cells was 74.21% and 72.15% in triangle region. But absolute number was still limited. There was no significant difference in morphology and growth using the two separating medium. Immunocytochemical staining showed four kinds of cells. The first type was C-kit＋ cell colony. The second type was cells positively expressing CD29^＋, CD90^＋, CD34^-, C-kit- （characteristics of mesenchymal stem cells） and slightly expressing cytokeratin 19 （characteristics of bile duct cells）. The third type was cells strongly expressing cytokeratin 19 and negatively expressing C-kit antigen. The fourth type was cells highly expressing CD45 and weakly expressing CD34, CD90 and C-kit. After induction, keratin 19 of interface cells was strongly expressed, but negatively expressed C-kit, albumin, alpha fetoprotein and cytokeratin 18. CONCLUSION： In the fetal liver, number of C-kit^＋ cells is few. Ficoll and Percoll could improve the harvest of C-kit^＋ cells derived from fetal liver. There is mesenchymal stem cells and duct cells type, and it can improve the growth of C-kit^＋ cells. Mesenchymal stem cells can differentiate into duct cells.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2009年第1期111-116,共6页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金资助项目(30771959)~~
