目的:研究骨髓间充质干细胞(MSCs)条件培养液对小鼠未成熟卵母细胞体外成熟的作用.方法:分离、培养小鼠MSCs,获得MSCs条件培养液.收集3类生发泡期卵母细胞,分别在对照培养基和条件培养液中培养,观察卵母细胞成熟率,判断最佳时间点；FDA、Hoechst33258和PI联合染色评价细胞活力；荧光标记检测皮质颗粒分布、迁移及纺锤体复合物的形成情况.结果:条件培养液组3类生发泡期卵母细胞的成熟率高于对照培养基组；其中,完全/大部分裸露的生发泡卵母细胞和周围有疏松的颗粒细胞包裹的生发泡卵母细胞的最佳体外成熟时间为16h,有完整的数层颗粒细胞紧密包裹的生发泡卵母细胞的最佳体外成熟时间为24 h.体外成熟卵母细胞活力良好,皮质颗粒分布及纺锤体复合物形成与体内成熟卵母细胞一致.结论:MSCs条件培养液有利于小鼠体外成熟卵母细胞核、细胞质同步成熟,提高卵母细胞质量,是一种较好的体外成熟培养体系.%Objective: To investigate the effects of conditioned medium (CM) of mesenchymal stem cells (MSCs) on the in vitro maturation of mouse immature oocytes. Methods: The mouse MSCs were isolated and cultured to harvest CM. Three different oocytes from immature germinal vesicle (GV) stage were collected and cultured in the basal medium (DMEM, Stempro) and CM (DCM, SCM) respectively. The optimal time and maturation rate of invitro maturation (IVM) were surveyed, Oocytes were stained with fluorescein diacetate(FDA), Hoechst33258 and prodium iodide(PI) to evaluate the cell viability. The behaviors of cortical granules (CG) and spindle complexes were examined under a fluorescence microscope. Results : Three categories of GV oocytes cultured in CM were all achieved a higher maturation rate than those cultured in basal medium. The optimal maturation time wre 16 h for oocytes only or surrounded by loose granulosa, while 24 h for oocytes surrounded by intact granulosa cells layers. Oocytes kept a good viability in CM culture condition during IVM. The distribution of CG and movement of spindle was complex in IVM oocytes, which were well consistent with in vivo matured oocytes and showed good markers of cytoplasmic maturation. Conclusion: Conditioned medium of MSCs is an efficient IVM system, which benefits nuclear and cytoplasm maturation of mouse IVM oocytes synchronously.
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