一种新的人胚胎干细胞无饲养层培养方法的建立  

Establishment of Feeder -Independent Culture Systems for Human Embryonic Stem Cells

作　　者：何丽娟[1] 习佳飞[1] 曾泉[1] 张博文[1] 杨超[1] 岳文[1] 裴雪涛[1] 

机构地区：[1]军事医学科学院野战输血研究所,北京100850

出　　处：《生物技术通讯》2010年第5期660-665,共6页Letters in Biotechnology

基　　金：国家高技术研究发展计划(2006AA02A107);北京市科委科技计划研发攻关类重大项目(D07050701350702)

摘　　要：目的:以转染碱性成纤维细胞生长因子(bFGF)的人胎肝基质细胞株(FLSC)培养人胚胎干细胞(hESC),寻找更加安全、有效的体外培养扩增方法。方法:通过ELISA方法定量检测转基因的人FLSC条件培养基中bFGF的分泌量;以商业化的mTeSR1无血清无饲养层培养基、常规小鼠胚胎成纤维细胞(MEF)条件培养基,以及转染bFGF的人FLSC条件培养基(bFGF/FLSC-CM)分别培养扩增H9细胞。通过观察hESC形态、免疫荧光染色、流式细胞检测及RT-PCR,检测hESC全能性标志物的表达。结果:ELISA方法检测bFGF/FLSC-CM中bFGF因子的分泌量为(770.09±17.28)pg/mL,而MEF-CM中bFGF因子的分泌量为(55.59±0.61)pg/mL,两者存在显著差异(P<0.01);在3种培养体系下,免疫荧光检测hESC全能性标志Oct-4、Tra-1-81抗体的表达均呈阳性,流式检测细胞表面阶段特异性胚胎抗原4(SSEA-4)抗体阳性细胞的比例均在99%左右;RT-PCR检测到hESC特异的转录因子Oct-4、Nanog、Sox-2的表达。结论:以转染bFGF的人FLSC条件培养基可以有效扩增hESC,可为临床应用提供一种安全、高效、低成本的无饲养层培养方法。Objective： To seek novel in vitro expansion methods to expand human embryonic stem cells（hESC） by transgenic human fetal liver stromal cells（FLSC） culture condition.Methods： Transgenic FLSC were measured secretion of basic fibroblast growth factor（bFGF） by ELISA.H9 cell lines were cultured with mTeSR1 feeder-independent culture medium,mouse embryonic fibroblasts（MEF） conditioned media,FLSC transfected with bFGF conditioned media,respectively.The cell morphology was followed up with an inverted microscope.The markers characteristic of hESC were analyzed by immunofluorescence and flow cytometry.Total cellular RNA was extracted by using TRIzol reagent followed by reverse transcription to detect Oct-4,Nanog,Sox-2.Results： The secretion of bFGF was significantly different between transgenic FLSC conditioned media and MEF conditioned media[（770.09± 17.28）pg / mL and（55.59±0.61）pg / mL,respectively].In three culture condition,immunofluorescence analysis showed an undifferentiated hESC colony with positive immunoreactivity for internal（Oct-4） or cell surface（Tra-1-81） markers.The percentages of stage-specific embryonic antigen 4（SSEA-4） positive cells were almost 99% by flow cytometry analysis.RT-PCR demonstrated hESC expressed transcription factors characteristic of hESC.Conclusion： Human FLSC transfected with bFGF conditioned media were one of the efficient and lower cost culture systems for feeder-independent hESC culture condition.

关 键 词：人胚胎干细胞 转染 人胎肝基质细胞 碱性成纤维细胞生长因子 

分 类 号：Q813[生物学—生物工程]