Objective To investigate the role of Nanog, one of the core genes regulating pluripotency of stem cells, in differentiation of primcrdial germ cells towards gonocytes. Methods The primordial germ cells were obtained from 11.0 dpc (days post coitus) rat fetuses, and they were cultured in vitro according to the criteria for embryonic stem cell culture, and transfected with Ihree designed small interfering RNA (siRNA) targeting Nanog gene by liposome. After tTansfection, the gene silence efficiency was assessed. RT-PCR and immunofluorescen.ee techniques were employed to detect Nanog expression on RNA level and protein level respectively. The typical embryonic stem cell-like colonies were counted under inverted microscope to evaluate the proliferation and nndifferentiation status of EG cells. The apoptosis of EG cells was detected by TLTNEL assay and transmission electron microscopy. Semi-quantitative RT-PCR assay was carried out to assess mRNA expression of gonosyte meiosis-associated marker genes (Mvh, Stra8, Sycp3), Resalts Transfection of siRNA efficiently inhibited the mKNA and protein expressions of Nraog within 48 hours. After 48h, the number of typical undifferentijted EG colonies decreased dramatically, while some EG cells presented differentiation signs and more suspending EG cells appeared. TUNEL assay and transmission electron microscopy revealed apoptosis of suspending EG cells. The mRNA expressions of Mvh, StraS and Sycp3 were up-regulated as detected by RT-PCR. Conclusions The molecular events that pluripotent primordial germ cells differentiate towards gonocytes and initiate meiosis after arriving gonids may be related to the down expression of core regulating gene Nanog of pluripotent stem cells.%目的 探讨多能干细胞核心调控基因Nanog在原始生殖纽胞(PGCs)向生殖母细胞方向分化过程中的作用.方法 取11.0d胎鼠PGCs,按胚胎干细胞(ES)培养标准进行体外胚胎生殖细胞(EG细胞)培养.采用脂质体方法将Nanog靶向特异性小干扰RNA(siRNA)转染人EG细胞.RT-PCR、免疫荧光法检测siRNA的沉默效率；在倒置相差显微镜下进行体外克隆计数,检测EG细胞增殖未分化状态；TUNEL法和透射电镜检测EG细胞凋亡情况；半定量RT-PCR检测与生殖母细胞减数分裂相关的标志基因(Mvh、Stra8、Sycp3)的mRNA表达.结果 转染siRNA后48h EG细胞Nanog mRNA和蛋白表达明显受抑,典型未分化EG细胞克隆数量显著下降,呈现分化现象,悬浮细胞增多.TUNEL法、透射电镜检测悬浮EG细胞呈现凋亡征象.伴随Nanog表达下调,Mvh、Stra8、Sycp基因mRNA表达上调.结论 到达生殖腺后,多能性PGCs发生生殖母细胞化,开始减数分裂,该过程可能与多能干细胞核心调控基因Nanog的表达下调有关.
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