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目的:建立C57BL/6J×129/J杂交小鼠ES细胞系。方法:收集3.5d.p.c.的囊胚,培养在预先铺有小鼠成纤维细胞(MEFs)的高糖DMEM培养液中。3-4d后,挑出内细胞团(ICM),消化后重新种到新鲜的有MEFS培养液中。等到有典型的ES样集落长出,即传代以得到永久ES细胞系。通过分析碱性磷酸酶活性,SSEA-1,Oct-4的表达和形成畸胎瘤的能力来鉴定ES细胞的多向分化能力。结果:获得的两个C57BL/6J×129/J杂交小鼠ES细胞系绝大多数细胞具有正常的核型(40,XY),碱性磷酸酶染色阳性,SSEA-1,Oct-4表达阳性,ES细胞注入SCID鼠后可获得来自3个胚层的组织。结论:建立了两株具有长期自我更新能力和多向分化潜能的C57BL/6J×129/J杂交小鼠ES细胞系。
Objective: To establish a C57BL / 6J × 129 / J hybrid mouse ES cell line. Methods: 3.5 d.p.c. blastocysts were collected and cultured in high glucose DMEM medium pre-plated with mouse fibroblasts (MEFs). After 3-4 days, the inner cell mass (ICM) was picked and re-seeded into fresh MEFS medium after digestion. When typical ES-like colonies grow, they are passaged to obtain a permanent ES cell line. The multidrug differentiation ability of ES cells was identified by analyzing alkaline phosphatase activity, the expression of SSEA-1, Oct-4 and the ability to form teratomas. RESULTS: Most of the ES cell lines derived from two crossed C57BL / 6J × 129 / J mice had normal karyotype (40, XY), positive for alkaline phosphatase, positive for SSEA-1 and Oct-4 , Tissue from 3 germ layers was obtained after ES cells were injected into SCID mice. Conclusion: Two C57BL / 6J × 129 / J hybrid mouse ES cell lines with long-term self-renewal ability and multidirectional differentiation potential were established.