兔骨髓基质细胞核移植的初步研究

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。

Preliminary research on nuclear transfer by using bone marrow stromal cells as nuclear donors in rabbits

Objective To establish a practical protocol for somatic cell nuclear transfer by using bone marrow stromal cells (BMSCs) in rabbits so as to culture pluripotent embryonic stem cells (ESCs) derived from inner cell mass (ICM) of cloned blastocysts. Methods Matured oocytes from superovulated rabbits were enucleated and inserted with single BMSCs into the perivitelline space. After electrofusion, the reconstructed couples were activated with ionomycin followed by 6-dimethylaminopurine (6-DMAP) + Cytochalasin B (CB). The cloned embryos were cultured in vitro in TCM-199 containing 10% fetal bovine serum (FBS). Results 147 oocytes were enucleated and injected with single BMSCs. The success rates of enucleation and injection were 70.75% (104/147) and 82.69% (86/104), respectively. Fusion rate of the couples was 56.79% (46/81). The rates of cleavage and blastocyst developed from the reconstructed embryos were 65.22% (30/46) and 17.39% (8/46), respectively. 62.5% (5/8) of blastocysts obtained from cloning were developed to hatching stage. Blastomeres expelled from zona pellucida adhered to the bottom of petri-dish and spread, which showed the potential developmental ability to the further stage. It makes the possibility to isolate and culture ESCs from ICM of cloned blastocysts. Conclusion It is feasible to isolate ICM cells from cloned blastocysts produced via nuclear transfer by using BMSCs as nuclear donors.