人胚胎肝脏细胞表型特征分析及肝脏前体细胞的克隆化培养  

The phenotypic characteristics of human fetal liver progenitors and clonal culture in vitro

作　　者：王萍[1] 李卫红[2] 刘保清[2] 路欣[3] 尤红[1] 贾继东[1] 张海燕[2] 

机构地区：[1]首都医科大学附属北京友谊医院肝病研究中心,北京100050 [2]首都医科大学细胞生物学系 北京市高等院校“肝脏保护与再生调节”重点实验室,北京 [3]首都医科大学人体解剖学与组织胚胎学系

出　　处：《中华肝脏病杂志》2010年第10期763-767,共5页Chinese Journal of Hepatology

基　　金：基金项目：国家自然科学基金（30971472）;北京市自然科学基金（5102013）

摘　　要：目的 研究体外胚胎期人肝脏细胞（FLCs）的表型特征并利用克隆培养法获得肝脏前体细胞.方法 用免疫荧光染色和流式细胞仪测定FLCs的抗原表达;采用克隆化培养法结合不同生长因子组合的培养基对FLCs进行单细胞克隆筛选,用克隆环分离各单克隆细胞,扩大培养后应用逆转录聚合酶链反应法测定不同克隆mRNA的表达.用SPSS12.0软件单因素方差分析不同培养基配方克隆形成率.结果 原代分离培养的FLCs呈非典型的上皮细胞形态、大小不均一,其肝脏前体细胞标志物甲胎蛋白、白蛋白、细胞角蛋白8和细胞角蛋白19的阳性率分别为28.1%、84.7%、55.1%和9.1%;造血干细胞标志物CD34和CD45的阳性率为0.04%和0.09%;间质细胞标志物CD105和CD166的阳性率分别为71.8%和75.3%.采用克隆化培养法获得的细胞克隆形态有一定差异,多数克隆由长梭形细胞组成,部分由形状不规则、胞体较铺展的细胞组成,少数克隆具有类上皮细胞形态.对可连续传代5次的19个细胞克隆进行RT-PCR分析结果显示,表皮细胞黏附分子、甲胎蛋白、白蛋白和细胞角蛋白19的阳性率分别为52.6%,21.1%,52.6%和84.2%.结论 原代培养的FLCs是混杂有多种类型细胞的异质性群体,克隆培养法可以获得肝脏前体细胞克隆.Objective To investigate the phenotypic characteristics of human fetal liver cells （FLCs） and to obtain the homogenous hepatic progenitors with cloning. Methods Immunofluorescence and flow cytometry were used to determine the phenotypes of the FLCs. The proliferating colonies were isolated using clone ring in different culture conditions. Reverse transcriptase-polymerase chain reaction （RT-PCR） was used to determine the mRNA expression after further cultivation. Results The cultured FLCs showed a nontypical epithelial morphology. The positive rate for hepatic cell specific markers alpha-fetoprotein （AFP）,albumin （Alb）, cytokeratin 8 （CK8） and CK19 were approximately 28.1%, 84.7%, 55.1% and 9.1% respectively.percents Furthermore, the FLCs expressed the hematopoietic stem cell markers CD34 and CD45 with percentages of 0.04% and 0.09%. 71.8% and 75.3% of the FLCs were positive for the mesenchymal cell marker CD105 and CD166. Most of the colonies showed an elongated morphology, some with an unregular spreading-out morphology, only a small number of colonies with an epithelial-like morphology. RT-PCR results showed that among the 19 colonies obtained after further cultivation and the percentages of epithelial cell adhesion molecule （EpCAM）, AFP, Alb and CK19 were 52.6%, 21.1%, 52.6% and 84.2%, respectively.Conclusions The clonal culture system is beneficial to obtain the homogenous hepatic progenitor cells from the heterogeneous culture of FLCs.

关 键 词：干细胞 克隆细胞 体外研究 细胞培养 肝脏前体细胞 

分 类 号：R686[医药卫生—骨科学]