人骨髓基质细胞核移植重构胚的建立

Establishment of reconstructed embryos by transferring nuclear of human bone marrow stromal cell into enucleated oocyte of rabbit

目的建立人体细胞核移植技术,为治疗性克隆的实验研究及临床应用奠定基础。方法应用显微操作技术将单个人骨髓基质细胞注入去核后的兔卵母细胞内,经电融合、离子霉素和6-甲氨基嘌呤激活后,培养于含10%胎牛血清的TCM-199中,使重构胚进一步发育至囊胚。结果245枚卵母细胞进行去注核显微操作,核移植后有72.6%(178/245)卵母细胞仍保持完整;经电融合后细胞融合率为62.8%(83/132);体外培养后约65%(54/83)的重构胚可进入分裂期,3.7%(2/54)发育至桑囊期,囊胚孵化率为100%(2/2)。结论兔卵母细胞能使人骨髓基质细胞核重新编程形成核移植重构胚并可继续发育至囊胚,使得从核移植重构囊胚分离胚胎干细胞用于实验和临床治疗研究成为可能。

Objective To establish the nuclear transfer technique by using human somatic cells as the nuclear donor cells for the experimental research and clinical application of therapeutic cloning. Methods Single human bone marrow stromal cell （hBMSC） was injected with micromanipulative technique into the perivitelline space of a enucleated oocyte of rabbit. After electrofusion and activation with ionomycin, 6-dimethylaminopurine （6-DMAP） and Cytochalasin B （CB）, the reconstructed embryos were cultured in vitro in TCM-199 containing 10% fetal bovine serum （FBS） for further development into the blastula. Results Totally 245 oocytes were enucleated and then accepted the new nuclear injection. The success rate of nuclear transfer was 72.6% （178/245） and fusion rate was 62.8% （83/132）. After cultured in vitro, 65% （54/83） of reconstructed embryos showed the division appearance, and 3.7% （2/54） of them developed into the morula. The hatching rate of reconstructed blastocysts was 100% （2/2）. Conclusion Rabbit oocyte can support hBMSC nuclear to re-program and form the reconstructed blastocyst. It shows the possibility to isolate and culture embryonic stem cells （ESCs） from inner cell mass （ICM） of reconstructed blastocysts for further research and clinical treatment.