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目的用解冻后废弃胚胎及部分患者捐献的冷冻胚胎建立胚胎干细胞系。方法用程序解冻法解冻2001-2007年来我生殖中心就诊的患者剩余冷冻胚胎,培养至囊胚,Pronase酶去除透明带后,全囊胚置于小鼠饲养层(MEF)中培养获得原代细胞,机械法结合Ⅳ型胶原酶消化法传代,并利用细胞形态观察、免疫荧光染色、核型分析、畸胎瘤切片胚层分析方法对所建胚胎干细胞系进行鉴定。结果序贯培养23枚废弃胚胎,有4枚培养到囊胚阶段;去除透明带后有2枚在MEF上贴壁生长,但只有1株成功扩增稳定传代至第80代;细胞集落呈典型的鸟巢状,有稳定的46XY核型;细胞系表达SSEA-3、SSEA-4、TRA-1-60和TRA-1-81抗原,RT-PCR检测有Nanog、Oct-4、Sox2等基因表达;同时在体外能分化为内、中、外3个胚层。结论解冻后2～3细胞Ⅲ～Ⅳ级的废弃胚胎仍具有发育形成囊胚的潜能,并利用所得囊胚采用酶去透明带全胚培养法能建立稳定增殖的hESC细胞系。
OBJECTIVE To establish embryonic stem cell lines with frozen embryos and some frozen embryos donated by some patients. Methods The frozen thawed embryos from patients who visited our reproductive center from 2001 to 2007 were thawed by program thawing method and cultured to blastocyst. After removing the zona pellucida with Pronase enzyme, the whole blastocysts were cultured in mouse feeder layer (MEF) to obtain primary cells , Mechanical method combined with type Ⅳ collagenase digestion method, and the use of cell morphology observation, immunofluorescence staining, karyotype analysis, teratoma section germ layer analysis of the constructed embryonic stem cell lines were identified. Results Twenty-three embryos were cultured in vitro and cultured in blastocyst stage. After removal of the zona pellucida, two of them were adherently growing on the MEF, but only one of them successfully propagated and grew steadily to the 80th generation. The cell colonies were typical . The cell lines expressed SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 antigen, and Nanog, Oct-4 and Sox2 genes were detected by RT-PCR ; At the same time in vitro can differentiate into three kinds of germ layer. CONCLUSION: The embryos with stages Ⅲ ~ Ⅳ in 2 ~ 3 cells after thawing still have the potential to develop into blastocysts. The hESC cell lines with stable proliferation can be established by using the enzyme-devoid zona pellucida whole-embryo culture.