单细胞克隆小鼠胚胎干细胞系的建立

目的:建立胚胎干细胞的培养系统及培养方法.方法:用免疫溶解法(immunosurgery)分离3.5 d的129SvJ系小鼠囊胚内细胞团,在γ射线照射的胎鼠成纤维细胞饲养层上培养,胰酶消化传代.用组织化学法、免疫组织化学方法识别细胞表面标志,常规G带法行染色体核型分析,在体、离体分别观察细胞的多向分化能力.结果:得到一个单细胞克隆129系小鼠胚胎干细胞系G11能长期稳定增殖不分化,具有正常40XY核型,碱性磷酸酶阳性、SSEA-1阳性,在体和离体条件下可分化为多种细胞.结论:以小鼠为模型成功建立了哺乳动物胚胎干细胞的培养方法和培养系统.

Establishment of a single cell-cloned mouse embryonic stem cell line

OBJECTIVE: To establish embryonic stem cells culture system and methods. METHODS: Inner cell masses were isolated from blastocysts of 3.5 day-old 129SvJ mice by immunosurgery, seeded onto gamma-irradiated mouse fibroblasts feeder layer. The outgrowths were passed by digestion with trypsin. Surface markers were identified by cytochemistry and immunohistochemistry. Karyotype was tested with standard G-banding technique. Differential potency was tested both in vivo and in vitro. RESULTS: One single cell cloned mouse embryonic stem cell line G(11) was established, which had normal 40XY karyotype and proliferated rigorously for a long time, expressed surface markers such as AP and SSEA-1, and was able to differentiate into many cell types both in vivo and in vitro. CONCLUSION: Mouse embryonic stem cell culture system has been established in our laboratory.