化学去核卵母细胞为受体的小鼠体细胞核移植

Reconstruction of Mouse Embryos with Chemically Enucleated Oocytes

【目的】本研究旨在探索一种崭新的、化学试剂诱导去核卵母细胞为核受体的、无透明带的、手工体细胞核移植方法。【方法】将第一次减数分裂期小鼠卵母细胞进行诱导去核并去除透明带,去核卵胞质与胎儿成纤维细胞粘合、电融合和SrCl2激活后,体外培养重构胚。【结果】重构胚融合率和激活率分别为84.8%和93.6%;胚胎2-细胞发育率为24.7%,4-细胞率为6.74%;2-细胞期克隆胚移植假孕受体后,没有获得怀孕受体;分别以"血清饥饿"胎儿成纤维细胞、新鲜细胞和冷冻保存细胞为供体作核移植,结果表明,冷冻保存细胞的融合率(69.3%)与其余两组(80.6%和84.8%)呈显著差异(P<0.05);激活率、2-细胞和4-细胞发育率,则3组间差异不显著(P>0.05)。【结论】本文将小鼠卵母细胞的化学去核与无透明带技术相结合,获得的克隆胚目前已发育到4-细胞期;另外,供体细胞的3种准备方式均不影响胚胎发育率。该方法属手工克隆,它的成功将会大大简化核移植程序,提高核移植总效率。

[ Objective] The objective of this sludy was to describe a handmade cloning method which combines the chemical induced enuleation and zona-free technology in embryo culture. [Method] Enuleated oocytes were derived by exposing the oocytes to demecolcine and cytoheximide supplemented mdium sequently and its chromosome was depleted to the first polar body. Then the zona and polar body of oocytes treated with drugs were removed by transferring into the M2 containing 0.5% protease. The mouse fetal fibroblast cells were glued to the zona-free oocytes membrane with phytohemagglutinin. Consequently the oocyte-cell couplets were fused by electronic pulse and the reconstructed embryos were transferred into M16 and cultured in well of well after activation with SrCl2 for 6 h. [Result] The rate of successful fusion was 84.8% and 24.7% of reconstructed embryo cleaved. Embryos in 4-cell stage were derived by this method （6.74%）. No pregnancy was observed after the 2-cell embryos were transferred to the surrogate mouse, Additionally, the effect of 3 protocols for donor preparation on the development of reconstructed embryos was studied. The results showed that there were no significant differences among groups except fusion rate（P〉0.05）. And thus the freeze preservation is a simple but efficacious protocol for preparing donor cells in nuclear transfer. [Conclusion] Further research is required to improve the developmental potential of reconstructed embryos produced by this new method and its availability will greatly facilitate the nuclear transfer in mammal.