骨髓间充质干细胞条件培养液对小鼠卵母细胞的孤雌激活作用

Effects of Conditioned Medium of Mesenchymal Stem Cells on Mouse Oocyte Parthenogenetic Activation

目的研究骨髓间充质干细胞(mesenchymal stem cell,MSC)条件培养液对小鼠MII卵母细胞的孤雌激活作用及胚胎发育能力。方法分离、培养小鼠MSC,获取MSC条件培养液(conditioned medium of MSC,CM)。通过促排技术获取小鼠MII卵母细胞,分别采用CM、7%乙醇、IVF方法激活,体视显微镜下观察原核形成及囊胚形成率。在CM激活后不同时间点,利用α/β-tubulin抗体标记纺锤体,激光共聚焦显微镜下观察有/无细胞松弛素B(CB)存在时纺锤体的运动变化。结果 CM可以激活小鼠MII卵母细胞,最佳刺激时间为40min,激活率达到95.4%,囊胚形成率为62%,与7%乙醇组比较无显著差异,但明显低于IVF组(95.4%vs.100%;62%vs.88%,P<0.01)。CB可以抑制纺锤体的旋转,阻止第二极体的排出,促进二倍体孤雌胚形成,提高囊胚形成率(62%vs.9%,P<0.01)。结论 CM能有效激活小鼠MII卵母细胞并促进孤雌发育。

Objective To investigate the effects of conditioned medium of mesenchymal stem cells （CM） on the parthenogenetic activation and development of mouse MII oocytes. Methods Mouse mesenchymal stem cells （MSC） were isolated and further cultured to collect CM. Mouse MII oocytes were collected with superovulation and activated with CM, 7% ethanol,or in vitro fertilization （ IVF）,respectively. Pronuclei formation and embryo development were evaluated under the view of a stereomicroscope. At various time-points after activation with or without the presence of cytochalasin B （CB）,the dynamic changes of meiotic spindle,as visualized in preparations stained for α/β-tubulin and chromatin,were observed by fluorescent confocal microscopy. Results CM effectively activated MII oocytes with the optimal time of 40 min. The rates of activation and blastocyst showed no significant difference between CM and 7% ethanol but were significantly lower in both compared with that in the IVF group （95. 4% vs. 100% ,P〈0. 01; 62% vs. 88% ,P〈0. 01）. CB inhibited the spindle rotation and polar body extrusion but did not affect chromosomal movement and nuclear division and,thus,facilitated the diploid formation and blastocyst rate （62% vs. 9% ,P〈0. 01）. Conclusion CM is an effective medium for mouse MII oocyte activation and subsequent embryo development.