慢病毒介导的稳定表达bFGF的胎儿肝脏基质细胞株的建立

Establishment of Fetal Liver Stroma Cell Lines Which Stably Express Basic Fibroblast Growth Factor by Lentiviral System

通过重组慢病毒系统感染人胎儿肝脏基质细胞(fetalliverstromalcell,FLSC),建立了能够稳定、高效表达碱性成纤维细胞生长因子(basicfibroblastgrowthfactor,bFGF)的细胞株bFGF/FLSC.从流产胎儿肝脏组织分离富集基质细胞,对其进行了生长特性和表面标志的鉴定,其在体外维持传代35代,依然保持正常的染色体核型.从胎儿骨髓间充质干细胞中克隆得到bFGF基因,构建重组慢病毒载体,感染FLSC,根据荧光表达强弱进行流式分选,获得能够继续稳定传代的低表达和高表达bFGF的两株细胞,RT-PCR和蛋白质印迹证实,细胞株中bFGF基因的稳定表达.RT-PCR结果显示,弱荧光和强荧光表达细胞的bFGF,在mRNA水平的表达分别是转染空载体细胞的2.33倍和6.19倍;蛋白质印迹结果显示,在蛋白质水平表达分别是1.76倍和5.05倍.用建立的bFGF/FLSC作饲养层细胞体外培养人胚胎干细胞(humanembryonicstemcells,hES),结果证明,其能在无或少量添加外源bFGF的条件下,维持人ES细胞增殖及其干性达20代.bFGF/FLSC细胞株的建立,将为构建低成本、安全高效的人胚胎干细胞的培养体系及研究造血细胞的发育分化提供适宜的微环境.

Basic fibroblast growth factor （bFGF or FGF-2） plays an important role in modulating proliferation, migration, differentiation and survival of various cells, such as fibroblasts, endothelial cells, smooth muscle cells and neural cells. The fetal liver stromal cell lines （FLSCs） expressing bFGF stably has been established by lentiviral system. The FLSCs were isolated from aborted fetus with informed consent, the growth character was identified by MTT method and the surface marker analyzed by flow cytometry. The cells could maintain normal karyotype after passaged for 35 generations. The coding region of 18 ku bFGF was cloned from fetal bone marrow mesenchymal stem cells （BMSCs） by RT-PCR. The bFGF recombinant lentiviral plasmid was steadily transfected into FLSCs. The low and high bFGF-expression FLSCs that transfected by the recombinant lentivirus were sorted by fluorescence-activated cell sorting （FACS） according to weak and strong eGFP expression. Analysis of weak and strong eGFP expression transfected FLSCs by RT-PCR and Western-blot demonstrated the stable expression of bFGF after 15 passages. The bFGF expression at mRNA level of weak and strong eGFP expression FLSCs are 2.33 and 6.19-fold for the FLSCs transfected by the control lentivirus, and then at the protein level are 1.76 and 5.05-fold. hES cells can be matained for 20 passages using the bFGF/FLSCs as feeder layers supplemented with a little or no additional recombinant bFGF. The establishment of bFGF/FLSCs could be acted as feeder cells for sustainment of human embryonic stem cell in vitro by supplying an eligible microenvirnment free of animal feeder layers.