人胚胎生殖细胞饲养层的制备  

The preparation of human embryonic germ cell feeder layer

作　　者：姜蓉[1] 黎晓莉[1] 吴宏[1] 王璐[1] 

机构地区：[1]重庆医科大学基础医学院组织学与胚胎学教研室干细胞与组织工程研究室,重庆400016

出　　处：《重庆医科大学学报》2011年第12期1440-1444,共5页Journal of Chongqing Medical University

基　　金：重庆市教委科学技术研究资助项目(编号:KJ090318)

摘　　要：目的:建立稳定高效的人包皮成纤维细胞(Human foreskin fibroblasts,HFF)饲养层培养体系,用于培养人胚胎生殖细胞(Embryonic germ cells,EGCs)。方法:取4～5岁小儿包皮,采用机械法和组织消化法分离、培养成纤维细胞,倒置相差显微镜观察细胞生长状况,免疫细胞化学法、MTT法、流式细胞术检测细胞纯度、生长曲线及细胞周期,筛选丝裂霉素C作用的最佳浓度和时间。分离5～11周人胚胎原始生殖细胞(Primordial germ cells,PGCs)并培养于经丝裂霉素C处理的HFF饲养层上,观察人EGCs生长情况。结果:机械法和组织消化法结合分离HFF方法可靠,纯度高,细胞生长活性好,能快速进入对数生长期,细胞周期与细胞生长曲线保持一致,细胞可以传60代以上,P3～P30代均适宜作饲养层;丝裂霉素C抑制细胞增殖的适宜浓度及时间为12.5 mg/L作用2.5 h;分离的人PGCs在该饲养层上培养可形成典型的EGCs集落,体外连续培养超过3代。结论:HFF饲养层能有效地支持人EGCs的生长。Objective：To establish a stable and effective system of human foreskin fibroblast（HFF） feeder layer in order to culture human embryonic germ cells（EGCs） in vitro.Methods：The HFF were isolated and cultured from the 4～5 year old children＇s foreskin through mechanical method and primary tissue digestion.The condition of cell growth was observed under inverted phase contrast microscope.The purity of cells,cell growth curve and cell cycle of HFF were detected by immunocytochemistry,MTT and flow cytometry.The optimal concentration of mitomycin C and the time for treating with mitomycin C on HFF were determined by MTT assay.The primordial germ cells from human embryos of 5～11 weeks were isolated;EGCs were cultured on the HFF feeder layer treated by mitomycin C and the state of cells growth was observed.Results：The mechanical method and primary tissue digestion was a credible and efficient way to obtain primary HFF,with which the cells had high purity and active proliferative abilities,and quickly entered exponential phase of growth.The cell cycle corresponded to cell growth curve.The fibroblasts,with an anticipated life span of 60 generations,were used in our experiments after 3～30 generations.HFF proliferation could be efficiently repressed after being treated with mitomycin C 12.5 mg/L for 2.5 h.Human EGCs cultured in the HFF feeder layer could form typical embryo germocyte colony and last P3 generations continuously.Conclusion：HFF feeder layer could efficiently support the growth of human EGCs.

关 键 词：人胚胎生殖细胞 包皮成纤维细胞 饲养层 

分 类 号：R321.1[医药卫生—人体解剖和组织胚胎学]