不同发育阶段人胚胎原始生殖细胞的定位及体外培养

Localization and in vitro culture of human embryonic primordial germ cells

目的 :对不同发育阶段的人胚胎原始生殖细胞 (PGC)进行定位并比较其体外培养的差异。 方法 :取 4～ 7周发育阶段的人胚胎 ,应用组织化学法检测PGC内碱性磷酸酶 (AP)以对其定位。体外分离人胚胎生殖嵴和肠系膜组织进行培养 ,第 1次传代后 ,用AP标记 ,计数不同发育阶段AP阳性的PGC和体外培养的阳性克隆并进行比较。 结果 :在卵黄囊、原肠、背侧肠系膜和生殖嵴处有AP强阳性细胞 ,阳性信号位于胞质内。阳性细胞散在分布 ,在生殖嵴处聚集成团。不同发育阶段的胚胎组织培养均有阳性克隆出现。统计学分析结果表明 ,4～ 7周人胚胎内AP阳性的PGC数目及细胞内信号强度无明显差异(P >0 .0 5 ) ;由胚胎组织培养获得的AP阳性克隆数也无显著差异。 结论 :人PGC位于卵黄囊、原肠、背侧肠系膜和生殖嵴处。 4～ 7周发育阶段 。

Objective: To study the localization and in vitro culture of human embryonic primordial germ cell(PGC) during different stages of embryogenesis. Methods: Human PGCs were detected by histological staining of alkaline phosphatase (AP) activity of different stages embryos from week 4 to 7.Human embryonic genital ridges and dorsal mesenteries were disaggregated and then cultured. Ten days after the first passage, AP activity detection was used as marker to identify positive clones. Primordial germ cells and clones with high level of AP activity were counted and the differences were estimated. Results: Human PGCs with AP were observed in yolk sac, primitive gut, dorsal mesenteries and genital ridges. AP staining signal was in cytoplasmic and kept steady during different embryos stages from week 4 to 7. AP PGCs scattered and aggregated in genital ridges; different stages of embryonic culture all had positive clones. Statistic analysis indicated no significant differences among primordial germ cells with AP, AP signal and positive clones during different stages of human embryogenesis. Conclusion: Human embryonic PGCs are localized in yolk sac, primitive gut, dorsal mesenteries and bilateral genital ridges. There are no significant differences among total primordial germ cells in vivo and clones when cultured in vitro during different stages from week 4 to 7 in human embryogenesis.