供体细胞来源和TSA处理对牦牛iSCNT胚胎重编程的影响

Effects of donor cell source and TSA treatment on reprogramming of yak-bovine iSCNT embryos

【目的】探讨不同来源的供体细胞和TSA处理对牦牛异种体细胞核移植(Interspecies nuclear trans-fer,iSCNT)胚胎的影响。【方法】以黄牛卵母细胞为受体,分别以牦牛50日龄胎儿及6和18月龄个体成纤维细胞为供体,构建牦牛iSCNT胚胎,研究供体细胞来源对牦牛iSCNT胚胎的影响。分别用0(对照),20,40,60和80nmol/L的TSA处理50日龄胎儿成纤维细胞12h,构建iSCNT胚胎,比较其发育情况,确定TSA的最佳处理浓度;用最佳浓度的TSA处理50日龄胎儿成纤维细胞0(对照),6,12和18h,构建iSCNT胚胎,比较其发育情况,确定TSA的最佳处理时间。用最佳TSA作用时间和浓度处理供体细胞,构建牦牛iSCNT,之后用40和80nmol/L的TSA分别处理iSCNT胚胎6和12h,比较不同处理方式对牦牛iSCNT胚胎发育的影响。采用qRT-PCR方法检测40nmol/L TSA处理供体细胞6h的iSCNT胚胎及40nmol/L TSA处理供体细胞6h且处理iSCNT胚胎12h的iSCNT胚胎去乙酰化酶2(HDAC2)基因mRNA的表达水平,以不进行TSA处理的iSCNT胚胎为对照。【结果】以50日龄胎儿成纤维细胞作为供体细胞,牦牛iSCNT胚胎的囊胚率最高,但与其他组差异不显著(P>0.05)。40nmol/L TSA处理供体细胞6h能显著提高牦牛iSCNT胚胎的囊胚率(30.6%),且与对照组差异显著(P<0.05)。用40nmol/L TSA处理牦牛iSCNT胚胎12h组的囊胚率高于80nmol/L TSA处理iSCNT胚胎6h组,2组间差异不显著(P>0.05)。用TSA处理供体细胞和iSCNT胚胎之后,HDAC2mRNA表达水平降低,且与对照组差异显著(P<0.05)。【结论】不同来源的供体细胞对iSCNT胚胎发育的影响不明显;40nmol/L TSA处理供体细胞6h和iSCNT胚胎12h能显著提高牦牛iSCNT胚胎体外发育及重编程能力。

【Objective】The present study was to investigate the effect of different sources of donor cells and TSA treatment on in vitro development of yak interspecies somatic cell nuclear transfer（iSCNT）embryos.【Method】With cattle oocytes as recipient,yak fetal fibroblasts of 50-day-old,6 and 18-month-old adult fibroblasts as the donor nuclear,yak iSCNT embryos were reconstructed and then the effect of donor cell source on the development of cloned embryos evaluated.We compared the effect of different TSA treated concentrations（0,20,40,60 and 80 nmol/L）and times（0,6,8 and 12 h）on the development of yak cloned embryos,respectively,then determined the best program for treatment,and treated donor cells and embryos with this program for detecting the effect of TSA on the relative expression level of HDAC2 by qRT-PCR.【Result】With fetal fibroblast cells as donor cells,yak interspecies cloning embryos blastocyst rate was the highest,but there was no significant difference（P〉0.05）.Treatment donor cell with 40 nmol/L TSA for 6 h significantly increased the blastocyst rate（30.6%）,and significant difference existed with the control group（P〈0.05）.Treatment of reconstructed embryos with 40 nmol/L TSA for 12 h was helpful for embryos development.However,the blastocyst formation rate decreased along with the treated time and concentration of TSA.The expression level of HDAC2 was reduced significantly after donor cell and embryos treated with TSA.【Conclusion】There was no significant effect of donor cells source on the development of yak iSCNT embryos.Treated donor cells for 6 h and reconstructed embryos for 12 h with 40 nmol/L TSA can significantly improve in vitro development of yak iSCNT embryos and reprogramming.