猪卵母细胞化学辅助手工去核及手工核移植胚胎发育能力因素的研究

Studies on Factors Affecting Chemical Assisted Handmade Enucleation of Porcine Oocytes and Developmental Capacities of Handmade Somatic Cells Nuclear Transfer Embryos

本研究在筛选猪化学辅助手工去核最佳脱羰秋水酰碱(Demecolcine,DC)浓度的基础上,探讨了各种因素对手工重构胚发育的影响,以期建立高效的猪手工体细胞核移植技术体系。观察不同成熟时间卵母细胞在不同DC浓度下的去核效率,并进一步比较了化学辅助手工去核法和荧光染色去核法、不同类型的供体细胞(颗粒细胞、新生巴马肌肉成纤维细胞和胎儿成纤维细胞)和供体细胞的不同处理方法(70%～80%汇合、血清饥饿法和100%接触抑制法)对重构胚发育能力的影响。结果表明:(1)利用DC诱导去核,对体外成熟培养44h的卵母细胞以浓度0.4μg.mL-1作用0.5～1h为宜。(2)用DC化学辅助手工去核与用荧光染色去核的重构胚囊胚发育率差异不显著(13.11%vs 9.25%,P>0.05)。(3)以胎儿成纤维细胞、颗粒细胞和新生巴马小型猪肌肉成纤维细胞为供体构建的重构胚的融合率、卵裂率及囊胚率均差异不显著(P>0.05)。(4)用70%～80%汇合(对照组)组、接触抑制组和血清饥饿组的细胞作供体构建的重构胚,在融合率上,接触抑制组显著高于对照组(P<0.05);在分裂率与囊胚率上,各组之间均无显著差异(P>0.05)。研究结果表明,化学辅助手工去核法在实际生产中可以替代荧光染色去核法,能省去去核过程中荧光染色及紫外光照射去核等步骤,从而简化了猪手工体细胞核移植程序,提高了猪手工体细胞核移植效率,能为高效的猪手工体细胞核移植技术体系的建立提供参考。

To optimize current handmade nuclear transfer technology of porcine,this study was conducted to investigate the effects of various factors on the developmental capacities of handmade somatic cells nuclear transfer（NT） embryos,based on screening optimal concentrations of demecolcine（DC） during the chemical assisted handmade enucleation of porcine oocytes.Firstly,the efficiency of handmade enucleation of porcine oocytes with different maturation time in DC with different concentrations has been investigated.Then,the effects of different enucleation methods,donor cells types（granule cells,fetal fibroblast cells and Bama mini-pig muscle fibroblast cells）,different treatment methods of donor cells（70%-80% contact inhibition as control,serum starvation,100% contact inhibition） on the developmental capacity of nuclear transfer embryos has been observed.The results showed：（1） The optimal treatment of DC for enucleation of porcine oocytes following maturation for 44 h in vitro was 0.4 μg·mL-1 for 0.5-1 h.（2） There was no significant difference in the rate of hand-made cloning（HMC） blastocyst developed from oocytes enucleated by DC assistance and the fluorescence staining group（13.11% vs 9.25%,P〉0.05）.（3） There was no significant difference in the developmental capacity among embryos reconstructed by using granule cells,new-born Bama mini-pig muscle fibroblast cells and fetal fibroblast cells as donor cells（P〉0.05）.（4） Among embryos reconstructed from donor cells of 70%-80% contact inhibition（control group）,100% contact inhibition and serum starvation,the fusion rate was significantly higher in 100% contact inhibition group than that in the control group（P〈0.05）.However,no significant difference was observed in the rate of cleavage and blastocyst among the three groups（P〉0.05）.These results imply that chemical assisted handmade enucleation of porcine oocytes in vitro can instead of ultraviolet light linked to a fluorescent dye method in practice,which can simplify the procedure of handmade somatic cells nuclear transfer in pig,thus provide the reference for establishing an efficient platform of hand-made somatic cell nuclear transfer in pig.