摘要
通过重组慢病毒系统感染人胎肝基质细胞(fetal liver stromal cells,FLSCs),建立了能够稳定高效表达促红细胞生成素(erythropoietin,EPO)的细胞株EPO/FLSCs.从胎儿肝脏克隆EPO基因,构建重组慢病毒EPO的表达载体,感染FLSCs,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高表达EPO基因的FLSCs,RT-PCR和ELISA结果证实,细胞株中的EPO基因稳定表达.RT-PCR结果显示,FLSCs的EPO在mRNA水平的表达分别是未转染FLSCs和转染空载体FLSCs的5.63倍和5.71倍.ELISA法检测了转染重组慢病毒EPO表达载体的FLSCs EPO蛋白表达水平,结果显示EPO蛋白的表达水平也明显升高.收集EPO/FLSCs的条件培养基,体外诱导脐血CD34+细胞向造血细胞分化,结果显示向红系定向分化的细胞比例明显居多,有可能为临床细胞治疗提供稳定、高质量的细胞来源.
Erythropoietin （EPO） plays an important role in modulating proliferation and differentiation of erythrocytes. The fetal liver stromal cell lines（FLSCs） expressing EPO has been established steadily by lentiviral system. The EPO gene was cloned from human fetal liver by RT-PCR. The EPO recombinant lentiviral plasmid was steadily transfected into FLSCs. The efficiency of virus transfection was identified by expression of enhanced green fluorescence protein（eGFP） analyzed by fluorescence microscope, then the high eGFP espression FLSCs were sorted by fluorescence-activated cell sorting （FACS） according to strong eGFP expression. Analysis of strong eGFP expression was detected by RT-PCR and ELISA. The EPO expression at mRNA level of strong eGFP expression FLSCs are 5.63 and 5.71-fold for the FLSCs no transfected and the FLSCs transfected by the control lentivirus. And at protein level, the content of EPO expression is 263 U/L. Then the supernatant from the EPO transfected FLSCs could induce the CD34＋ cell differentiated into hematopoietic cell, especially erythrocytes. This would provide an alternative for cell therapy and blood cell transfusion.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2010年第4期381-388,共8页
Progress In Biochemistry and Biophysics
基金
国家高技术研究发展计划(863)重大专项(2006AA02A107)
国家重点基础研究发展计划(973)(2005CB522702)
北京市科委科技计划研发攻关类(D07050701350702)资助项目~~
