昆明鼠胚胎干细胞系建立中原代克隆生长及其传代方法的研究

Study on the primary clonies culture and passage of embryonic sterm cells from Kunming mouse

目的:探讨昆明鼠胚胎内细胞团分离方法、原代干细胞克隆分离时机及干细胞传代方法对干细胞建系效率的影响。方法:取昆明鼠3.5d囊胚建胚胎干细胞系,比较全胚培养法与免疫外科法两种内细胞团分离方法对胚胎干细胞建系效率的影响,观察原代克隆的分离时机,机械法传代和酶消化传代对胚胎干细胞建系效率的影响。结果:全胚培养法30个囊胚建系3个,免疫外科法32个囊胚建系4个,两组均可以有效地形成胚胎干细胞系;昆明鼠原代干细胞克隆分离最佳时机是增殖4～6d;5代以前的干细胞以机械传代方法较好,5代后用酶消化法传代效果较好。结论:全胚培养法和免疫外科法均能有效建立昆明鼠胚胎干细胞系,采用机械化与酶消化法相结合传代更适合昆明鼠干细胞建系。

Objective： Try to investigate the separation method of the inner embryonic cell mass of Kunming mouse, separation time of primary colonies and the effect of passage method on the efficiency of stem cell establishment. Method ： The cell line of embryonic stem cells was established from Kunming mouse blastocysts at fetal age 3.5 day. The affect of the establishment efficiency was compared between intact embryo cultivation and immunosurgery for the isolation of the inner cell mass. The separation time of the primary clone was observed. The affect of the establishment efficiency was compared between mechanical method passage and enzymatic digestion passage. Results ： We established 3 cell lines of embryonic stem cells after 30 blastocysts were cultured by intact embryo cultivation. We established 4 cell lines of embryonic stem cells after 32 blastocysts were treated by immunosurgery. Both two groups can establish embryonic stem cell lines effectively. The best time for the isolation of the Kunming mouse primary stem cell clones is 4 ～ 6 d proliferation. Mechanical method is better for the stem cells before 5 generations, and enzymatic digestion passage method is better for the stem ceils after 5 generations. Conclusion： Both intact embryo cultivation method and immunosurgery method can effectively establish Kunming mouse embryonic stem ceil lines. The passage with the combination of mechanical and enzymatic digestion methods is more suitable for the establishment of embryonic stem ceil from Kunming mouse .