绵羊胚胎成纤维细胞饲养层用于培养人诱导性多能干细胞的效果研究

Research on the Effect of Sheep Embryonic Fibroblast Feeder for Culturing Human Induced Pluripotent Stem Cell

试验在体外分离培养1月龄绵羊胚胎成纤维细胞(sheep embryonic fibroblast,SEF),经丝裂霉素C处理后探讨其作为诱导性多能干细胞(induced pluripotent stem cells,iPSC)体外培养饲养层的可行性。试验以SNL饲养层细胞为对照,人iPSC(hiPSC)为培养对象,通过形态学观察、碱性磷酸酶(AP)染色、以及实时荧光定量PCR和免疫细胞化学对hiPSC标志基因mRNA和蛋白表达的检测,比较了SEF细胞和SNL细胞作为干细胞饲养层的效果。结果表明,在试验期内,与SNL饲养层体外培养的hiPSC相似,SEF饲养层体外培养的hiPSC在形态上呈集落样生长,增殖速度快;AP染色呈蓝紫色,能够维持未分化状态;能正常表达多能性标志基因。两种饲养层细胞培养的hiPSC多能性标志基因c-Myc、Klf4、OCT 4和SOX 2 mRNA的表达以及OCT4、SOX2、SSEA4和TRA-1-60蛋白的表达并无显著差异(P>0.05)。本研究结果初步表明SEF可作为体外培养iPSC的饲养层细胞,为进一步建立可表达促生长因子的基因修饰SEF细胞系奠定了基础。

The aim of this study was to investigate the feasibility of using the isolated and mitomycin C-treated sheep embryonic fibroblast(SEF)in vitro as the feeder layer of the induced pluripotent stem cells(iPSCs).Taking SNL feeder layer cells as the control group and the human iPSC(hiPSC)as the experimental object,the effects of SEF cells and SNL cells as feeder layer of stem cells were compared by the morphological observation,the alkaline phosphatase(AP)staining,and the qRT-PCR and immunocytochemistry detection of the mRNA and protein expression for hiPSC marker genes.The results demonstrated that,being similar to hiPSC on SNL feeder layer,hiPSC on SEF feeder layer showed rapid growth and proliferation as colonies during the experimental period.Meanwhile,the AP staining was blue-purple,the undifferentiated state was maintained,and the pluripotent marker genes were normally expressed.The expression of hiPSC pluripotent marker genes c-myc,Klf4,OCT4,and SOX2 mRNAs,as well as the expression of OCT4,SOX2,SSEA4,and TRA-1-60 proteins were not significantly different for hiPSCs on SEF and SNL(P>0.05).The results indicated that SEF could be used as feeder layer cells for culturing iPSC in vitro,which laid a foundation for the further establishment of the gene-modified SEF cell lines that could express the growth-promoting factors.