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目的通过建立骨髓基质细胞核移植的方法,为进一步从克隆囊胚的内细胞团细胞分离培养出全能性的胚胎干细胞并用于治疗性克隆等研究奠定基础。方法超排获得的兔成熟卵母细胞去核后,将同种的单个骨髓基质细胞核注入其内;电融合后,经离子霉素(ionomycin)和6-甲氨基嘌呤(6-DMAP)激活,用10%胎牛血清(FBS)的TCM-199进行体外培养直至囊胚阶段。结果实验对147个卵母细胞去核,去核成功率为70.75%(104/147);注核后有82.69%(86/104)的卵细胞保持形态完整;重构后的体细胞-卵母细胞质复合体经电融合后的融合率为56.79%(46/81);激活后的重构胚于体外培养后分裂率为65.22%(30/46),囊胚率为17.39%(8/46),获得的囊胚有62.5%(5/8)进入孵化阶段;孵化出来的细胞贴壁生长并向周围扩展,显示了具有向下一阶段发育的潜能,为从囊胚内细胞团细胞中分离并培养出胚胎干细胞提供了可能。结论实验结果表明,以兔成体体细胞的骨髓基质细胞为核供体,经克隆技术生产出囊胚并从中分离内细胞团细胞具有可行性。
OBJECTIVE: To establish a foundation for further research on cloning of pluripotent embryonic stem cells from the inner cell mass of cloned blastocysts by establishing bone marrow stromal cell nuclear transfer. METHODS: The mature oocytes obtained from superovulation were enucleated and then injected into the same single bone marrow stromal cell nucleus. After electrofusion, they were activated by ionomycin and 6-methylaminopurine (6-DMAP) Culture in vitro with TCM-199 with 10% fetal bovine serum (FBS) up to the blastocyst stage. Results 147 oocytes were enucleated and the success rate of enucleation was 70.75% (104/147). After the injection, 82.69% (86/104) of oocytes remained intact. The reconstructed somatic cell - oocyte The fusion rate of the cytoplasm complex after electrofusion was 56.79% (46/81). The activated embryos were 65.22% (30/46) in vitro and the blastocyst rate was 17.39% (8/46) ), 62.5% (5/8) of the blastocysts obtained entered the incubation stage; the hatched cells grew adherently and expanded to the periphery, showing the potential to develop to the next stage, It is possible to isolate and culture embryonic stem cells. Conclusion The experimental results show that it is feasible to produce blastocysts by cloning technology and to isolate the inner cell mass from the bone marrow stromal cells of rabbit somatic cells.