单链DNA结合蛋白2对小鼠胚胎干细胞向滋养层方向分化的作用

Effects of single-stranded DNA binding protein 2 on trophoblastic differentiation in mouse embryonic stem cells

目的寻找并鉴定可促进小鼠胚胎干细胞(ES细胞)向滋养层方向分化的新基因。方法优化小鼠ES细胞向滋养层方向分化的诱导条件,结合数据分析寻找在此分化过程中表达上调的基因,由此发现了单链DNA结合蛋白2(Ssbp2)。通过qRT-PCR检测Ssbp2在小鼠ES细胞向滋养层方向分化过程中的表达变化,并检测Ssbp2在小鼠早期胚胎对应的3种细胞类型中的表达模式。克隆Ssbp2基因并将其在小鼠ES细胞中过表达,观察细胞形态并检测其是否具有促进小鼠ES细胞向滋养层方向分化的功能。结果通过尝试不同的组合,发现在无血清的小鼠滋养层干细胞(TS细胞)基础培养液中添加BMP4联合b FGF既可诱导小鼠ES细胞中滋养层标记基因的显著上调,又可有效抑制中胚层标记基因的上调。通过表达模式分析发现,在诱导小鼠ES细胞向滋养层方向分化过程中Ssbp2的表达量发生了显著上调,并且Ssbp2在TS细胞中高表达。当在小鼠ES细胞中过表达Ssbp2之后,ES细胞发生了一定程度的分化,并且滋养层相关的标记基因发生了最为显著的上调。结论 Ssbp2具有促进小鼠ES细胞向滋养层方向分化的作用。

Objective To identify novel genes promoting trophoblastic differentiation in mouse embryonic stem（ES） cells.Methods Inducing conditions for trophoblastic differentiation in mouse ES cells were optimized.Combined with data analysis,significantly up-regulated genes during this differentiation process were identified and single-stranded-DNA binding protein 2（Ssbp2） was discovered as a candidate gene involved in trophoblastic differentiation.Changes in Ssbp2 expression during the process of trophoblastic differentiation in mouse ES cells,as well as expression patterns of Ssbp2 in three cell types corresponding to early mouse embryos,were detected by qRT-PCR.Then Ssbp2 genes were cloned and overexpressed in mouse ES cells.Cell morphology was observed and whether Ssbp2 genes could promote trophoblastic differentiation in mouse ES cells was detected.Results Through trials with different combinations,we found serum-free mouse TS cell basic medium with BMP4 and bFGF could not only induce significant up-regulation of trophoblast markers in mouse ES cells,but also efficiently repress the up-regulation of mesoderm markers.Expression pattern analysis showed that the expression level of Ssbp2 was significantly up-regulated during the process of inducing trophoblastic differentiation in mouse ES cells and Ssbp2 expression was high in mouse trophoblastic TS cells.After overexpression of Ssbp2 in mouse ES cells,ES cells were partially differentiated and the up-regulation in trophoblastic markers was most significant.Conclusion Ssbp2 can promote trophoblastic differentiation in mouse ES cells.