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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。
OBJECTIVE: To culture human embryonic germ cells in vitro using human foreskin fibroblasts as feeder layer and observe the effect of feeder layer on their growth and to identify human embryonic germ cells. Methods Fetal fibroblasts from 4 to 5 years old were isolated and cultured in vitro. P3 to P30 offspring were inactivated by mitomycin and then plated. The secretion of fibroblasts was detected by enzyme-linked immunosorbent assay (ELISA) (FGF). Cultured human embryonic germ cells were cultured on human foreskin fibroblast feeder cells without any cytokines for 5 ~ 11 weeks. Human embryonic germ cell The activity of phosphatase was detected by immunocytochemistry. The expression of SSEA-1 and SSEA-4 was detected by immunocytochemistry. The expression of Oct-4 was detected by RT-PCR. Results Fibroblasts could be passaged for more than 60 passages and suitable for feeder layer from P3 to P30. FGF content in supernatant from P3 to P30 on fibroblasts was (172.09 ± 2.66) pg / L ~ (245.25 ± 1.66) pg / L between the fluctuations. Isolation and culture of human embryonic germ cells can form typical embryonic germ cell colonies on the feeder layer. After cultured in vitro for more than 3 generations, the alkaline phosphatase activity of the cultured human embryonic germ cells was strongly positive. The detection of undifferentiated colonies showed SSEA-1, SSEA-4 Positive, Oct-4 expression was positive. Conclusion Human foreskin fibroblasts as feeder layer can support the growth of human embryonic germ cells and maintain the undifferentiated state.