体细胞来源及培养代数对核移植重构胚发育的影响(英文)

The Effects of Different Donor Cells and Passages on Development of Reconstructed Embryos

为探讨体细胞来源及培养代数对核移植重构胚发育的影响 ,实验采用电融合法将小鼠 2 细胞胚胎卵裂球、胚胎干细胞 (ES)、胎儿成纤维细胞、耳成纤维细胞、尾尖成纤维细胞、睾丸支持细胞和精原细胞以及不同培养代次的胎儿成纤维细胞进行了核移植。结果显示 :2 细胞胚胎卵裂球供核重构胚发育最好 ,囊胚率为 7 4 %;ES细胞重构胚虽然发育率低 ,但仍有囊胚出现 ,比例为 0 7%;胎儿成纤维细胞重构胚最高发育阶段为桑椹胚 ,比例为0 2 %;精原细胞重构胚只能发育到 8 细胞阶段 ,比例为 0 3%;其他几类细胞重构胚则仅能发育至 4 细胞阶段。不同培养代数的胎儿成纤维细胞重构胚除第 3代外都可发育到 8 细胞阶段 ,且发育率差异不显著 ,但第一代细胞重构胚 2 细胞发育率 (4 0 7%)显著低于 2、3和 4代细胞重构胚。结果表明 :不同分化程度的细胞核移植后 ,重新编程的难易程度是不一样的 ,分化程度越高则重新编程越难 ;未调整细胞周期的ES细胞由于多数处于S期 ,所以重构胚发育率很低 ;体外培养传代有利于体细胞核移植后重新编程。

In order to study the effects of different donor cells and passages on development of nuclear transfer embryos,we constructed embryos by electrofusing several kinds of donor cells into enucleated MⅡ oocytes from Kun Ming (KM) mouse.These cells include 2-cell embryonic blastomeres,KMW embryonic stem (ES) cells,fetal fibroblast,ear fibroblast,tail tip fibroblast,sertoil cells and spermatogonia.Meanwhile,we compared the effects of passage numbers of fetal fibroblast cells on developmental competency after nuclear transfer.We found that 7.4% of reconstructed embryos from 2-cell embryonic blastomeres and 0.7% from ES cell could develop to blastocyst in vitro;embryos from fetal fibroblast could only develop to morula stage with the rate of 0.2%;embryos from spermatogonia could only develop to 8-cell stage and the rate was 0.3%;embryos respectively from ear fibroblast,sertoli cell and tail tip fibroblast could only develop to 4-cell stage.Although 2-cell development rate of embryos reconstructed from fetal fibroblast in first passage was significantly lower than those from the 2nd,the 3rd and the 4th passage,embryos from different passages could develop to 8-cell stage except the 3rd passage.The result indicated that it is more difficulty for terminally differentiated cell nuclei to be reprogrammed in enucleated MⅡ oocytes than for low differentiated cell nuclei.The reason of low development rate from ES cells maybe that most of ES cells was at S stage of the cell cycle,which out of coordination with MⅡ oocytes.We could conclude that culture and passage of donor cells might be benefit to nucleus reprogramming.