包皮成纤维细胞作为饲养层对人胚胎生殖细胞生长的影响

目的 以人包皮成纤维细胞为饲养层体外培养人胚胎生殖细胞,观察饲养层对其生长的影响,并对人胚胎生殖细胞进行鉴定。 方法 分离培养4～5岁小儿包皮成纤维细胞,取P₃～P₃₀代细胞用丝裂霉素灭活后铺板备用,酶联免疫吸附测定（ELISA） 双抗夹心法检测其分泌物成纤维细胞生长因子（FGF）的含量;分离培养5～11周人胚胎原始生殖细胞,在不添加任何细胞因子的人包皮成纤维细胞饲养层上培养人胚胎生殖细胞;细胞化学法检测人胚胎生殖细胞碱性磷酸酶的活性,免疫细胞化学法检测其胚胎表面特异性抗原SSEA-1及SSEA-4的表达,RT-PCR法检测Oct-4的表达。 结果 包皮成纤维细胞可以传60代以上,P₃～P₃₀代均适宜作饲养层;P₃～P₃₀代包皮成纤维细胞上清液中FGF含量在(172.09±2.66)pg/L～(245.25±1.66)pg/L之间波动。分离培养的人胚胎生殖细胞在饲养层上可形成典型的胚胎生殖细胞集落,体外连续培养超过3代,其碱性磷酸酶活性呈强阳性,集落未分化标志检测显示SSEA-l、SSEA-4呈阳性,Oct-4表达阳性。 结论 用人包皮成纤维细胞作为饲养层能支持人胚胎生殖细胞的生长并维持未分化状态。

Effect of human foreskin fibroblast as a feeder layer on growth of human embryonic germ cells

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