【摘要】 目的利用化学激活的技术,建立小鼠孤雌胚胎干细胞系。方法用钙离子载体A23187和6-二甲基氨基嘌呤(6-DMAP)处理小鼠卵母细胞,待囊胚形成后,分离内细胞团,细胞传代、扩增。待传至37代时,核型检测查染色体,用微卫星技术进行纯合性鉴定,用免疫荧光技术检测胚胎干细胞表面标记Oct-4、SSEA-1及SSEA-4的表达情况,并用畸胎瘤试验检测其多向分化能力。结果该细胞株为孤雌来源,表现为正常二倍体核型,表达ALK,表面标志物Oct-4、SSEA-1阳性,SSEA-4阴性。体内注射后,在局部形成含三个胚层组织的畸胎瘤。结论通过钙离子载体A23187和6-DMAP化学激活小鼠卵母细胞,可以得到具有胚胎干细胞特性的细胞株。更多还原

【Abstract】 Objective To creat a mouse parthenogenetic stem cell line by chemical activation. Methods Mouse oocytes were treated with A23187 and 6-DMAP, and then cultured till blastula embryos formed. Then the inner cell mass (ICM) cells were isolated, cultured, and passaged. Cells of the 37th passage were examined by karyotyping to analyze chromosomes, microsatellite technique to determine homozygous genotype, immunohistochemistry to detect expression of specific cell markers, and teratogenic test to exhibit multi-lineage potency. Results The cells had a homozygous genotype and normal karyotype. The cells were positive for alkaline phosphatase, Oct-4, and SSEA-1, and negative for SSEA-4. After injected into the SCID mice, the cells differentiated into three germ layers, and formed teratoma at the injection site. Conclusion An embryonic stem cell line can be created by A23187 and 6-DMAP activation of mouse oocytes.更多还原