胎儿成纤维细胞的培养分离方法对猪体细胞核移植胚胎发育潜力的影响

Influence of Different Culture Treatment Methods of Fetal Fibroblasts on Embryos Developmental Capacity of Porcine Somatic Cells Nuclear Transfer

研究采用不同传代的培养方法和不同方法处理及不同培养分离法探讨胎儿成纤维细胞的处理方法对猪体细胞胚胎发育潜力的影响。结果表明:(1)100%长满汇合胎儿成纤维细胞的核移植融合率高于70%～80%的胎儿成纤维细胞(P<0.05),分裂率高于血清饥饿培养的胎儿成纤维细胞(P<0.05),但囊胚率差异不显著(P>0.05);(2)猪胎儿成纤维细胞冷冻-解冻后的核移植分裂率和囊胚发育率均显著低于新鲜和4℃冷藏的细胞(P<0.05),但融合率无显著差异(P>0.05),表明猪胎儿成纤维细胞解冻后不宜直接进行核移植;(3)采用组织块法和酶消化法分离得到的胎儿成纤维细胞所构建的重构胚胎在融合率、分裂率和囊胚率方面没有显著差异(P>0.05)。表明100%长满汇合培养是较好的猪胎儿成纤维细胞培养处理方法;猪胎儿成纤维细胞解冻后不宜直接进行核移植;组织块法和酶消化法均可用于分离培养猪体细胞核移植的胎儿成纤维细胞。

Influences of different passage culture＇methods,different treatments methods and different cell dissociate culture methods of fetal fibroblasts on embryos developmental capacity of Porcine Somatic Cells Nuclear Transfer were mainly discussed in present research.The results showed：（1）The fusion rate in group of 100%contact and confluence was higher than one of the 70～80% contact group（P〈0.05）.The cleavage rate of fetal fibroblast in 100% contact and confluence group was higher than one of serum starvation group（P〈0.05）.Blastocyst rates in groups were of no significant difference（P〉0.05）.（2） The cleavage rate and the blastocyst rate of reconstructed embryos by using fetal fibroblasts in routine digestion group and 4℃frozen group were markedly higher than the frozen and thawed group（P〉0.05）.Fusion rates among three groups were not significantly different（P〉0.05）.The results showed that fetal fibroblasts were not suitable as donor cells after the freezing and thawing treatment.（3）By using culture method of tissue explant and enzymatic digestion,the developmental capacity of reconstructed embryos was discussed.The result also showed that the fusion rate,the cleavage rate and the blastocyst rate of the reconstructed embryos in fetal fibroblasts were not significantly different（P〈0.05）.In conclusion,100% contact and confluence is better for culture of fetal fibroblasts in porcine.It is not optimum for using thawed fetal fibroblasts to do Nuclear Transfer directly in porcine.The culture method of tissue explant and enzymatic digestion can both be used to dissociate and culture fetal fibroblasts in Somatic Cell Nuclear Transfer in porcine.