马羊膜上皮细胞分离、培养及影响因素的研究

Study on the isolation,culture,and influencing factors of equine amniotic epithelial cells

为了建立马羊膜上皮细胞(equine amniotic epithelial cells,e AECs)的分离、培养方法,并对其影响因素进行研究,试验从胎盘剥离羊膜,采用Trypsin-乙二胺四乙酸(EDTA)或Tryp LE消化法分离获得上皮细胞,然后进行上皮细胞培养传代研究。结果表明:通过0.25%Trypsin-EDTA消化,每克羊膜平均获得2.2×106个细胞,其3代传代倍增时间平均为(1.32±0.21)d;通过0.25%Tryp LE消化,每克羊膜平均获得1.9×106个细胞,其3代传代倍增时间平均为(1.26±0.17)d,两者间差异显著(P<0.05)。Tryp LE消化结果比较稳定,更适合从马羊膜层消化、分离上皮细胞。

To establish the methods to isolate and culture equine amniotic epithelial cells （eAECs）, and conduct the research on its influencing factors. The equine amniotic membrane was isolated from equine placenta, and then the eAECs were obtained using Trypsin - ethylene diamine tetra - acetic acid （EDTA） or Trypsin - like enzyme （TrypLE） digestion, and then conducted the research of culture and passage tot the eAECs. The results showed that 2.2 × 10^6 cells were on average acquired from 1 g of amniotic membrane using 0.25% Trypsin - EDTA diges, tion, and the doubling time of three - generation passage was on average （ 1.32±0.21 ） d; 1.9 × 106 cells were on average obtained from 1 g of amniotic membrane using 0.25 % TrypLE digestion, and the doubling time of three - generation passage was on average （ 1.26 ±0.17 ） d ; there was a significant difference （ P 〈 0.05 ） between the two groups. The results indicate that the result of digestion using TrypLE is more sta- ble, whic is suitable for the digestion and isolation of the eAECs from equine amniotic membrane.