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目的探讨以人胚胎成纤维细胞为饲养层分离、培养人胚胎生殖细胞的方法和条件。方法分离、培养3～4月胚胎成纤维细胞,取3～15代细胞经丝裂酶素处理后铺板备用;分离6～11周胚胎原始生殖细胞,将其置于人胚胎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;用免疫细胞化学方法检测胚胎生殖细胞表面标志SSEA1和SSEA4;钙钴法检测碱性磷酸酶活性;RT PCR检测转录因子Oct4的表达。结果人胚胎成纤维细胞可连续传代25代以上(6月),3～15代细胞可以用作饲养层细胞。分离的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8代。集落未分化标志检测显示SSEA1、SSEA4呈阳性,碱性磷酸酶活性呈强阳性,Oct4表达阳性。结论用人胚胎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 methods and conditions for the isolation and culture of human embryonic germ cells using human embryonic fibroblasts as feeder layer. Methods The fibroblasts from March to April were isolated and cultured. The cells of passage 3 to passage 15 were treated with mitomycin and then plated. The primordial germ cells from 6 to 11 weeks were isolated and placed on the feeder layer of human embryonic fibroblasts The embryonic germ cells were cultured in culture system containing growth factor and differentiation inhibitor. The surface markers SSEA1 and SSEA4 of embryonic germ cells were detected by immunocytochemistry. The alkaline phosphatase activity was detected by calcium and cobalt method. The transcription factor Oct4 expression. Results Human embryonic fibroblasts can be passaged for more than 25 generations (June), and cells of passage 3 to 15 can be used as feeder cells. Isolated embryonic germ cells can proliferate to form typical embryo germline colonies on the feeder layer and can be continuously cultured in vitro for more than 8 generations. Colonies undifferentiated markers showed SSEA1, SSEA4 positive, alkaline phosphatase activity was strongly positive, Oct4 expression was positive. Conclusion Human embryonic fibroblasts can be used as feeder layer to obtain continuous proliferative embryonic germ cells.