关键词: 包皮成纤维细胞, 胚胎生殖细胞, 饲养层, 酶联免疫吸附测定-双抗夹心法, 免疫组织化学, 反转录-聚合酶链式反应, 人

Abstract: Objective To culture human embryonic germ cells (EGCs) on feeder layer of human foreskin fibroblast (HFF), and observe the growth of EGCs and identify EGCs. Methods To separate and culture HFF of children aged 4-5, the fibroblasts from third generation to 30th were used in the experiment. The fibroblast growth factor(FGF) concentration in HFF culture medium was detected by ELISA-double antibody sandwich assay. The primordial germ cells were isolated from 5-11 weeks human embryos. The EGCs were cultured in medium without exogenous growth factors, only supported by HFF as feeder layers．The activity of alkaline phosphatase, specific antigen of the embryo SSEA-1 and SSEA-4 and Oct-4 were respectively detected by cytochemistry, immunocytochemistry and RT-PCR. Results The fibroblasts, with an anticipated life span were of 60 generations, were used in our experiments after 3-30 generations. The concentrations of FGF in HFF culture medium were ranged from (172.09±2.66) pg/L to (245.25±1.6) pg/L. Human EGCs cultured in the HFF feeder layer can form typical embryo germocyte colonies. After having been cultured for P3 generations continuously, the activity of alkaline phosphatase of EGCs, undifferentiation marker SSEA- and SSEA- of EGCs colonies and the expression of transcription factor Oct- showed positive. Conclusion HFF can be a feeder layer to support the growth of human EGCs and maintain its undifferenti

Key words: Foreskin fibroblast, Embryonic germ cell, Feeder layer, ELISA-ouble antibody sandwich assay, Immunohistochemistry, RT-PCR, Human