摘要
目的:探讨胚芽干细胞的提取、培养和体外扩增的最佳条件;筛选胚芽干细胞在体外培养中的活力最佳时期。方法:分离受精11d小鼠生殖腺嵴,经消化制备成胚芽干细胞悬液,将细胞分别接种于基础培养液、同种胎鼠成纤维细胞条件培养液、添加LIF的基础培养液、有饲养层的培养基和与胎鼠成纤维细胞共培养5种不同的培养体系中,比较观察在5种培养体系中胚芽干细胞的形态学、碱性磷酸酶染色和体外分化情况。结果:①在含15%胎牛血清的DMEM/F12基础培养液中培养时,胚芽干细胞贴壁明显减少,EGC克隆形成很少,传代率很低;②在基础培养液中添加白血病抑制因子(LIF)或同种胎鼠成纤维细胞条件培养液时,细胞生长情况与基础培养液培养相比无明显差异;③有饲养层存在时,4～6d后出现鸟巢状胚芽干细胞集落。传4代后集落逐渐减少、变小。6～7代后集落消失;④与胎鼠成纤维细胞共培养时,2～4d即可见EGC集落形成。传6代后细胞的集落逐渐减少、变小;⑤在与胎鼠成纤维细胞共培养时,第2、3代细胞的生长曲线基本相同,接种24h后细胞呈对数生长。培养液的更新可以显著影响小鼠胚芽干细胞的生长。第5代的细胞增殖速度减慢。结论:与饲养层细胞或腹壁组织成纤维细胞共培养可较好地培养和扩增胚芽干细胞;第3代细胞的活力最好。
Objective： To select the optimal growth method for the proliferation of embryonic stem cells from primordial germ cells in vitro. Methods： The gonadal ridges of the mouse embryos at 11days postcoitum （dpc） from Kunming female mice mated with Kunming males were isolated and disaggregated by 0.25% trypsin and 0.02% EDTA, then the embryonic germ cells （EGCs） were cultured respectively in DMEM/F-12 medium, in the conditioned medium from primary embryonic fibroblasts （PEF-CM）, in DMEM/F-12 with LIF （1 000 U/ml）, on the primary embryonic fibroblast feeders with the treatment of mitomycin, and co-cultured with the fibroblasts from the abdominal wall tissue of the same age murine fetus. The cell growth curves were tested in different generation under the condition of co-culture with the fibroblasts. Results： Without fibroblast feeder it was difficult for EGCs to grow well in DMEM/F-12, PEF-CM and DMEM/F-12 medium with LIF （1 000 U/ml）. The embryonic germ cells could proliferate on the fibroblast feeders treated with or without mitomycin. Both the clone number and the passage rate of EGCs co-cultured with fibroblasts isolated from the fetal abdominal wall tissue were higher than those of EGCs cultured on the feeder treated with mitomycin. EGCs in 3^rd generation proliferated fast. Conclusion： Co-culture with fibroblasts or culture on the fibroblast feeder cells treated with mitomycin are the optimal methods for EGCs to proliferate in vitro.
出处
《山东大学学报（医学版）》
CAS
北大核心
2005年第7期561-566,共6页
Journal of Shandong University:Health Sciences
基金
山东省科技厅攻关计划项目(01BB1DADA1)。
关键词
胚芽干细胞
体外培养
小鼠
Embryonic germ cells
Cuture in vitro
Mice
