鸡胚胎原始生殖细胞的培养和传代

Cultivation and passage of chicken primordial germ cells

本文旨在探索鸡胚胎原始生殖细胞(Primordialgermcells,PGCs)培养、传代以及各种因素对PGCs培养的影响。实验结果表明,在添加10%的胎牛血清、2%的鸡血清、2mmol/LL谷氨酰胺、1mmol/L丙酮酸钠、5.5×10-5mol/Lβ巯基乙醇、10μl/ml非必需氨基酸、以及5ng/ml人干细胞生长因子(Humanstemcellfactor,hSCF)、10U/ml鼠白血病抑制因子(Mouseleukemiainhibitoryfactor,mLIF)、10ng/ml碱性成纤维生长因子(Fibroblastgrowthfactorbasic,bFGF)、0.04ng/ml人白细胞介素11(Humaninterleukin11,hIL11),10ng/ml胰岛素样生长因子(Humaninsulinlikegrowthfactor,hIGF)的高糖DMEM培养体系中,以多次离散法进行传代获得5-6代鸡胚胎PGCs,有效地维持了PGCs的未分化状态和正常二倍体核型,同时能够定向地诱导分化为神经细胞。

This paper investigates proper conditions and factors for the cultivation and cryopreservation of chicken primordial germ cells （PGCs）. PGCs were isolated from germinal ridges at the 19th chick embryo stage with 0.25% trypsin-0.04% EDTA at room temperature cultured in the DMEM with high concentrations of glucose supplemented with 10% fetal bovine serum, 2% chicken serum, 1 mmol/L glutamine, 5.5×10^-5 β-mercaptoethanol, 100U/ml gentamycin and 1 mmol/L sodium pyruvate, with or without hSCF （5 ng/ml）, mLIF （10 U/ml）, bFGF （10 ng/ml）, hIL-11 （0.04 ng/ml）, IGF （10 ng/ml）. After three passage, chicken embryonic fibroblast cells were used as a feeder layer. Cell morphology, colony formation, proliferation capability, karyotype, alkaline phosphatase activity and cell differentiation were characterized. Results showed that the Medium Ⅱ which wassupplemented with 5 factors was better than the Medium Ⅰ which was without the factors. PCGs cultured in the Meium Ⅱ could be subcultured five times and still maintained an undifferentiated status with normal chromosome karyotype, the cytoplasm of PCGs exhibited positive reaction for PAS and alkaline phosphatase. Chicken primordial germ cells could be induced into neuron type cells by vitamin A. It seems that the cultured chicken PGCs maintained characters of pluripotent stem cells [ Acta Zoologica Sinica 51 （4）： 723-731, 2005].