鸡胚胎生殖细胞在鼠胚成纤维细胞饲养层上的生长

Growth of Chicken Embryonic Germ Cells on Mice Embryonic Fibroblasts

目的:探讨以鼠胚成纤维细胞为饲养层分离、培养鸡胚胎生殖细胞的方法和条件。方法:分离、培养12.5～13.5d鼠胚成纤维细胞。分离孵化5.5d鸡胚原始生殖细胞,原代培养时不使用饲养层,与性腺基质细胞共培养;继代培养时将其置于鼠胚成纤维细胞饲养层上,在含生长因子、分化抑制因子的培养体系中培养胚胎生殖细胞。结果:鼠胚成纤维细胞可连续传代18代以上(4个月),3～15代细胞可以用作饲养层细胞。分离的鸡胚胎生殖细胞在饲养层上可增殖形成典型胚胎生殖细胞集落,并能连续在体外培养超过9代。集落未分化标志高碘酸希夫反应(PAS)呈强阳性,体外分化实验表明胚胎生殖细胞具有多能性。结论:用鼠胚成纤维细胞作为饲养层能获得可连续增殖的胚胎生殖细胞。

Objective： To investigate the isolation, purification and culture methods of chicken embryonic germ cells （EGCs） on feeder layer cells of mice embryonic fibroblasts（MEFs）. Methods： Mice embryonic fibroblasts from the mice embryos of 12.5-13.5 days were cultured for over 18 passages（4 months）. Numerous primordial germ cells colonies were derived from gonadal ridges of 5.5-day-old chicken embryos. At primary culture, chicken primordial germ cells（PGCs） were co-cultured with the gonadal stromal cells. For subculture the PGCs were plated with mitotically inactive feeder layer cells of mice embryonic fibroblasts. The medium composed of growth factors and differentiation inhibitory factors. Results： The cultures had some characteristics of embryonic germ cells, such as the ability to be continuously passed（passage 15） and the morphology of periodic acid-Shift reaction（PAS） positive. The cultures would differentiated into several kinds of cells under the condition of devoid of LIF and feeder layer cells, such as fibroblast cells, neurocytes, autorhythmic cells, etc. When cultured in suspention, they successfully formed embryoid bodies. All of which indicate that the cells obtained were EGCs. Conclusion： Continuously proliferating EGCs can be obtained on feeder layer cells of MEFs.