包皮成纤维细胞的生物学特性研究

Biologic characters of human foreskin fibroblasts

目的：探讨人包皮成纤维细胞的生物学特性，为人胚胎生殖细胞（Human embryonic germ cells．hEG cell）长期体外培养打下基础。方法：探索分离包皮成纤维细胞的方法；采用免疫细胞化学法、MTT法、流式细胞术等对人包皮成纤维细胞进行生长形态观察，纯度鉴定，生长曲线及细胞周期检测，并就不同浓度丝裂霉素作用不同时间对人包皮成纤维细胞生长的影响进行研究。结果：0．25％胰酶，4℃消化过夜（12～16h）较37℃，5％CO2孵育2h更容易分离表皮和真皮；包皮成纤维细胞波形蛋白染色呈强阳性，纯度达95％以上；细胞生长曲线没有明显的滞留期，在1-6d处于快速增殖期，第7d开始处于平台期；细胞周期大多数处于Go／G，期，细胞周期与细胞生长曲线保持一致。丝裂霉素抑制包皮成纤维细胞增殖的适宜浓度及时间为12．5ug／ml作用2．5h。结论：包皮成纤维细胞的生物学特性表明其可以作为人EG细胞长期体外培养的饲养层。

Objective：To study the biologic characters of human foreskin fibroblasts（HFF） to lay the foundation for a long-term culture for human embryonic germ cells in vitro. Methods：The method of separating HFF was investigated. By immunocytochemical method,MTT and flow cytometry,we investigated the morphology and the purity of HFF,study the cell growth curve and cell cycle of HFF,and the effects of different concentrations of mitomycin on growth of HFF at different times. Results：Epidermis and dermis were more easily separated after overnight digestion（12-16 h） in 0.25% Trypsin at 4℃,compared with incubation for 2 h in 5% CO2 at 37℃. Vimentin was strongly positive stained with the purity of 95 percent. There was no obvious stranded period. The rapid proliferation period was in 1-6 d of cell growth curve. The platform stage began at 7 d. Majority of the cells were in G1/G0 phases. The cell cycle corresponded to cell growth curve. Mitomycin C（MMC） inhibited the proliferation of HFF at 12.5 ug/ml over 2.5 h of MMC treatment. Conclusion：hs biological characters showed that HFF can be used as feeder layer for in-vitro cultured human embryonic germ cells.