大鼠原生殖细胞培养和分化的研究

STUDY OF THE CULTURE AND DIFFERENTIATION OF PRIMORDIAL GERM CELLS IN RATS

研究大鼠胚胎原生殖细胞 (prim ordial germ cells,PGCs)的培养及分化。取受精后 11～ 12 .5天大鼠 PGCs进行原代培养 ,光、电镜观察 PGCs及其分化细胞的微细结构 ,碱性磷酸酶染色检测细胞的分化程度。结果显示大鼠 PGCs大而圆 ,散在分布 ,或多个聚集成团 ,胞质中含有椭圆形的线粒体和丰富的核糖体 ,在鼠胚成纤维细胞饲养层存在的情况下 ,PGCs保持未分化状态 ,碱性磷酸酶反应呈强阳性。在缺乏饲养层的条件下 ,PGCs很快分化 ,形态不规则 ,有伪足 ,碱性磷酸酶反应减弱 ,进一步分化可形成具有细长突起的神经元样细胞、胞质中含有细丝束的表皮细胞、可见节律性跳动的心肌细胞、具有分泌颗粒的分泌细胞及似血管、心脏形状的管腔结构等 ,由 PGCs分化来的细胞碱性磷酸酶反应均呈阴性。结果表明大鼠 PGCs能够分化形成三个胚层的衍生物 ,生殖嵴来源的 PGCs是一种具有发育全能性的胚胎多能干细胞。本研究同时证明鼠胚饲养层能抑制大鼠

The differentiation of primordial germ cells (PGCs) was studied in SD rats. Gonadal ridges of rat embryos 11 to 12 5 days postcoitum were disaggregated and cultured in different conditions. The morphology of undifferentiated and differentiated PGCs was observed using light and electron microscopy and the differentiality of PGCs was detected with alkaline phosphatase (ALPase) reaction. In the presence of feeder layer prepared from the primary culture of mouse embryos, PGCs were scattered or formed large compact multicellular colonies. Undifferentiated PGCs appeared as large and round cells, contained spherical mitochondria and abundant ribosomes, and expressed high level of ALPase activity. However, in the absence of feeder layer, PGCs were round or elongated in shape, with low reactivity of ALPase and pseudopodia were often present. PGCs differentiated into neuron like cells with long processes, epidermic cells with filaments, cardiomyocyte like cells with rhythmic beating, secretory cells containing secretory granules, and tubular structures similar to heart and vessels. These differentiated cells were ALPase negative. These results demonstrate morphologicaley and histochemcaley that rat PGCs have the pluripotentiality to differentiate in vitro into derivatives of three embryonic germ layers, and PGCs cultured on feeder layer of mouse embryos are undifferentiated.