马羊膜上皮干细胞的分离培养和鉴定

Isolation,culture and identification of equine amniotic epithelial cells

通过Tryp LE消化马羊膜收集羊膜上皮细胞,经过传代培养后,用免疫荧光染色、RT-PCR及流式细胞技术(FACS)分析其干细胞特征。免疫荧光染色发现马羊膜上皮细胞表达SSEA-1、SSEA-3、SSEA-4、TRA-1-60和TRA-1-81,只有少量的半贴壁细胞表达Oct4;RTPCR进一步证实马羊膜上皮细胞表达Oct4、Nanog和Sox2,而不表达STAT-3;流式细胞技术分析显示马羊膜上皮细胞也表达CD44、CD90、CD105,但不表达CD45。马羊膜上皮细胞培养三代时,平均倍增时间为(1.25±0.17)d。研究结果表明,通过Tryp LE消化马羊膜上皮可以分离获得马羊膜上皮细胞。

Equine amniotic membrane was digested with Tryp LE. The stem cells characteristic of equine amniotic epithelial cells were assessed by immunofluorescence technique,RT- PCR and flow cytometric analysis. Immunofluorescence assay showed that SSEA- 1,SSEA- 3,SSEA- 4,TRA- 1- 60 and TRA- 1- 81 could be expressed,and Oct4 was only expressed in a small amount of half- adherent cells. Expressions of Oct4,Nanog and Sox2,but not STAT- 3,were confirmed in the equine amniotic epithelial cells by RT- PCR. The flow cytometric analysis showed that CD44,CD90 and CD105,but not CD45,were also expressed in the cultured cells. The doubling time of the cultured cells was 1. 25 ± 0. 17 days on average within three passages. These results indicate that equine amniotic epithelial cells can be successfully obtained by digestion of amniotic membrane with Tryp LE.