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目的:利用长期冷冻人卵裂期胚胎经序贯共培养形成的囊胚建立人类胚胎干细胞系。方法:将冷冻≥10年的卵裂期胚胎复苏后,采用单层卵丘细胞序贯共培养至囊胚期,经辅助孵出,置鼠胚胎成纤维细胞(MEF)饲养层全胚培养,原代培养6d后加入20%MEF条件培养基培养至形成原代克隆。观察人胚胎干细胞集落的生长状态并通过碱性磷酸酶染色、Oct-4基因表达、核型分析及体外分化实验等方法进行生物学鉴定。结果:9枚冷冻卵裂期胚胎经序贯共培养,有6枚发育到囊胚期,最终获得1株胚胎干细胞系。经鉴定该细胞系具有碱性磷酸酶活性、表达Oct-4胚胎干细胞特异标记、形成拟胚体、在体外形成具有自动节律性的心肌细胞、并具有46,XY核型等5种特性。结论:冷冻胚胎经序贯共培养能够改善胚胎发育潜能,有助于建立人胚胎干细胞系。
OBJECTIVE: To establish a human embryonic stem cell line using blastocysts formed by sequential co-culture of long-term frozen human cleavage stage embryos. Methods: The cleaved embryos frozen more than 10 years after resuscitation, single-layer cumulus cells were co-cultured to the blastocyst stage, assisted hatch, set the mouse embryonic fibroblasts (MEF) feeder layer of whole embryo culture, Six days after primary culture, 20% MEF conditioned medium was added to form primary clones. The growth of human embryonic stem cell colonies was observed and their biological characteristics were identified by alkaline phosphatase staining, Oct-4 gene expression, karyotype analysis and in vitro differentiation assay. RESULTS: Nine cryogenic cleavage stage embryos were sequentially co-cultured, with 6 developing to blastocyst stage and 1 embryonic stem cell line finally obtained. The cell line was identified as having alkaline phosphatase activity, expressing Oct-4 embryonic stem cell-specific markers, forming embryoid bodies, and forming cardiomyocytes with automatic rhythmicity in vitro. CONCLUSIONS: Sequencing co-culture of frozen embryos improves embryonic development potential and facilitates the establishment of human embryonic stem cell lines.