原始生殖细胞(primordial germ cells,PGCs)是一类具有分化潜能的生殖细胞.为了检测激活素A(activin A)对鸡胚血液PGCs体外存活和增殖的影响,本研究从第13～15期鸡胚血液中分离提取PGCs,并进行鉴定;在完全培养液中分别添加0和100 ng/mL activin A培养48 h后,检测PGCs数目、形态及细胞周期;检测Dazl (deleted in azoospermia-like)基因甲基化水平;并通过Western blot检测转化生长因子-β(transforming growth factor-β,TGF-β)信号通路中TGF-β、SMAD2/3的表达和磷酸化变化.结果显示,从鸡胚血液中分离的PGCs,经过红细胞裂解液处理和传代后,可获得高纯度PGCs,且具有良好的生长趋势.培养的PGCs呈阶段特异性胚胎抗原(stage specific embryonic antigen 1,SSEA-1)、鸡(Gallus gallus)VASA同源基因(chicken vasa homologue,CVH)免疫染色和过碘酸希夫(periodic acid schiff reaction,PAS)反应染色阳性,并且表达多能性相关基因PouV、Nanog和Sox2 (SRY (sex determining region Y)-box 2),表明经过体外培养后,鸡胚血液PGCs仍能维持其生物学特性.添加activinA,可显著降低PGCs中Dazl基因甲基化水平,提高PGCs的体外存活能力.activinA处理后,显著增加了PGCs的数量;上调细胞周期蛋白(cyclins)及细胞周期蛋白依赖性激酶(cyclin-dependent kinases,CDKs)的表达;流式细胞检测进一步证实了activinA增加S/G2期PGCs细胞的比例.activin A显著上调了TGF-β蛋白的表达,促进SMAD2/3的磷酸化.以上结果表明,activinA可通过激活TGF-β/SMAD信号通路显著提高PGCs的体外存活和增殖能力.研究结果为揭示activinA对鸡胚PGCs增殖的作用机制提供了依据,为利用PGCs体外培养模型进一步深入研究生殖细胞发育调控提供基础资料.%Primordial germ cells (PGCs) are kind of cells which have the potential to differentiate into germ cells.To investigate the effect of activin A on the survival and proliferation of chicken (Gallus gallus) embryonic blood PGCs cultured in vitro,PGCs were isolated from stage 13～15 chicken embryonic blood,and then the cultured PGCs were identified using PGCs marker antibodies and marker genes.After addition of activin A (0 and 100 ng/mL) into complete medium for 48 h,the cell number,morphology and cell cycle were detected.To study the regulative mechanism of activin A in PGCs proliferation,the DNA methylation level of deleted in azoospermia-like (Dazl) gene was measured.The protein expression and phosphorylation of transforming growth factor-β (TGF-β) and SMAD2/3 were tested by Western blot.The results showed that PGCs isolated from chicken embryonic blood was distinct from blood cells with big nuclear and rich in glycogen particles.The purity of PGCs rapidly rised by treatment with red blood cell lysis buffer (ACK),from (0.026±0.005)％ to (69.2±4.6)％.The high purity and excellent growing trend of PGCs was obtained by subculture.After subculture for 3 passages,PGCs was positive with stage specific embryonic antigen 1 (SSEA-1),chicken vasa homologue (CVH) and periodic acid Schiff reaction (PAS).The expression of pluripotent related genes,PouV,Nanog and sex determining region Y (SRY)-box 2 (Sox2),suggested that the biology characterist theics were well maintained in PGCs cultured in vitro.After treated with activin A,numbers of PGCs were significantly increased and the morphology of PGCs was better than that of control group,with clear boundaries and cubic morphology.And the mRNA expression of cyclin-dependent kinases (CDK2)/yCclin D1 and CDK6/Cyclin E were obviously up-regulated.Flow cytometry analysis confirmed that PGCs populations treated with activin A displayed a significant increase in the proportion of S and G2 phase cells,which meant that there were more PGCs which entered into mitosis process than that of control group.The survival and proliferation ability of PGCs was enhanced by addition of 100 ng/mL activin A.Moreover,the ratio of Dazl gene mythelation was decrcascd from 94.7％ in control group to 52.6％ in activin A treated group.However,the mRNA expression of Dazl was significantly increased.The results indicated that activin A promoted PGCs proliferation by reducing the mythelation of Dazl gene.The protein expressions of TGF-β and SMAD2/3 were up-regulated and the phosphorylation of SMAD2/3 was activated when PGCs were treated with activin A.Collectively,these results suggested that activin A significantly promoted the survival and proliferation of PGCs cultured in vitro via TGF-β/SMAD signaling pathway and Dazl mythelation.These data provides basic information for the mechanism of PGCs proliferation regulated by activin A,and provides theoretical foundation to regulate germ cell development by using PGCs culture model.
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