目的：探索一种简单高效的分离、培养小鼠原始生殖细胞（PGCs）的方法，为小鼠模型提供充足的PGCs来源。方法体外分离小鼠12.5 dpc胚胎生殖嵴，剪碎后用含血清、胰岛素-转铁蛋白-硒添加剂、卵泡刺激素、重组表皮生长因子的α-M EM培养基进行组织块贴壁培养；于倒置显微镜下观察细胞形态，采用流式细胞术检测阶段性特异性胚胎抗原，采用实时荧光定量聚合酶链反应（RT-PCR）、细胞免疫荧光等方法检测干细胞的多能性基因Oct4及减数分裂Ⅰ期的几个特异性基因的表达。结果体外分离培养的小鼠 PGCs具有良好的细胞形态、极强的增殖能力，SSEA-1表达阳性，SSEA-4表达阴性，同时检测到Oct4、Stra8、Vasa、SCP3、ZP3表达均为阳性。结论利用该实验方法能培养出高纯度、具有良好干性及极强增殖能力的小鼠PGCs ，并使该细胞进入减数分裂。%Objective To explore a simple and efficient method for isolating and culturing mouse primordial germ cells (mPGCs) in vitro in order to provide the sufficient mPGCs sources of mouse model .Methods The 12 .5 dpc mouse embryonic gen-ital ridge were isolated in vitro and cut into pieces ,then the organization to adherent culture was performed in a-MEM medium con-taining fetal calf serum and insulin-transferrin-selenium (ITS) and follicle stimulating hormone(FSH) and recombinant endothelial growth factor(rEGF) .The inverted microscope was used to observe the cellular morphology .Flow cytometry was used to identify the stage specific embryonic antigens of cultured cells .Reverse transcription-polymerase chain reaction(RT-PCR) and immunofluo-rescence were used to identify the pluripotency gene Oct4 expressions of stem cells and specific genes in the meiosis phaseⅠ .Results The in vitro isolated and cultured mPGCs showed good cell morphology ,extremely strong proliferation capacity and the positive expression of SSEA-1 and negative expression of SSEA-4 ,Oct4 ,meanwhile the expressions of Stra8 ,Vasa ,Scp3 ,Zp3 were detected to be positive .Conclusion Using this culture method can culture the high purity of mPGCs with the excellent stem cell properties and extremely strong proliferative ability ,moreover which makes the cells entering the meiosis stage .
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