摘要
目的建立人胚胎干细胞体外培养模式,并对其进行冻存及复苏。方法将人胚胎干细胞置于小鼠胚胎成纤维细胞饲养层上培养,细胞接近融合状态时进行传代,程序降温法冻存,速溶方法复苏,确定解冻后细胞复苏率、克隆生长与分化情况,并对其生物学特性进行鉴定。结果人胚胎干细胞稳定增殖了5个月,传至20代;细胞在小鼠胚胎成纤维细胞饲养层上呈集落生长,核大,核仁明显。程序降温法冻存后复苏,细胞复苏率达到78.3%;解冻后第12代细胞仍具有稳定的46,XX核型;TRA-1-81阳性,表达Nanog基因,流式细胞术检测SSEA-4阳性表达率为89.38%。结论成功建立了人胚胎干细胞体外培养模式,为研究人胚胎干细胞及相关疾病的防治提供细胞模型和细胞来源。
Objective： To establish the culture mode of human embryonic stem （hES） cells in vitro and study the methods of cryopreservation and resuscitation. Methods： HES cells were cultured on mouse embryonic fibroblast （MEF） cells and were passaged when cell fusion. They were cryopreserved by programmed freezing method and were resuscitated by quick thawing. The recovered rates of frozen clumps, the levels of proliferation and differentiation of hES cells after freezing, thawing and culture were determined. The bio- logical characteristics of hES cells after thawing were identified. Results： HES cells were stable proliferation for 5 months and 20 passages. The cells accumulated as clumps on MEF feeder layer, which had large nuclei and transparent nucleoli. The recovered rate after programmed freezing was78. 3%. The passage 12 of programmed freezing hES cells retained normal 46, XX karyotype, expressed TRA - 1 -81 protein and nanog gene. Expression rates was 89. 38% of SSEA -4 by flow cytometry. Conclusion： The culture mode of hES cells in vitro was established successfully, which provide cell model andresource for the study of hES cells and the prevention , therapy of related diseases.
出处
《中国优生与遗传杂志》
2008年第2期17-19,61,共4页
Chinese Journal of Birth Health & Heredity
基金
全军十一五医药卫生科研重大课题(06G101)
关键词
人胚胎干细胞
体外培养
程序降温法冷冻
复苏
Human embryonic stem cell
Culture
Programmed cryopreservation
Resuscitation
