论文部分内容阅读
目的探讨体外分离培养人原始生殖细胞的条件和方法。方法分离、培养2～3个月人胚胎的成纤维细胞,经60Co照射后作为饲养层;从5～9周人胚胎的生殖嵴和肠背系膜组织中分离原始生殖细胞,培养在含有bFGF、LIF和Forskolin的人胚胎成纤维细胞饲养层上;同时采用免疫细胞化学和RT-PCR方法检测人原始生殖细胞阶段特异性胚胎抗原SSEA-1、SSEA-4以及特异性基因Oct-4、Ifitm-3、stella、Mvh、DAZL的表达,并进行类胚体培养。结果分离的人原始生殖细胞在饲养层上可以增殖形成典型的鸟巢状集落;细胞染色体均为正常的二倍体核型;阶段特异性胚胎抗原SSEA-1、SSEA-4表达阳性,同时检测到生殖干细胞特异性基因Oct-4、Ifitm-3、stella、Mvh、DAZL表达。人原始生殖细胞在悬滴培养时能够形成类胚体,表明其具有多项分化潜能。结论用人胚胎成纤维细胞作为饲养层进行体外培养人胚胎生殖嵴和肠背系膜组织,分离的原始生殖细胞形成EG细胞集落,初步鉴定为人胚胎生殖细胞。
Objective To investigate the conditions and methods for the isolation and culture of human primordial germ cells in vitro. Methods Fibroblasts of 2 ~ 3 months human embryos were isolated and cultured as feeder layer after 60Co irradiation. Primordial germ cells were isolated from the reproductive ridges and intestinal dorsal mesangial tissues of human embryos from 5 to 9 weeks and cultured in medium containing bFGF , LIF and Forskolin on the human embryonic fibroblast feeder layer. Simultaneous immunocytochemistry and RT-PCR were used to detect the SSGE-1, SSEA-4 and the specific genes Oct-4, Ifitm -3, stella, Mvh, DAZL expression, and embryoid body culture. Results The isolated human primordial germ cells proliferated into typical bird’s-nest colonies on the feeder layer. The chromosomes were normal diploid karyotypes. The positive expression of stage-specific embryonic antigens SSEA-1 and SSEA-4 was also detected Germline-specific gene Oct-4, Ifitm-3, stella, Mvh, DAZL expression. Human primordial germ cells are capable of forming embryoid bodies upon hanging drop culture, indicating that they have multiple differentiation potential. Conclusion Human embryonic fibroblasts are used as feeder layer to culture human embryonic germ ridge and intestinal dorsal mesenchyme. The isolated primordial germ cells form the colonies of EG cells and are initially identified as human embryonic germ cells.