优化山羊体细胞核移植方案的研究

以乳腺细胞、胎儿成纤维细胞和颗粒细胞为供体细胞进行核移植，比较了供体细胞类型、细胞冷冻及核移植的重建方法（胞质内注射、电融合）对重构胚早期发育的影响。结果：（1）乳腺细胞支持重组胚的发育能力（囊胚率7．14％）显著低于成纤维细胞（16．19％）和颗粒细胞（19．01％）（P〈0．05），卵裂率无显著差异（P〉0．05）；（2）乳腺上皮细胞构建重组胚时电融合法的效率（69．52％）极显著高于胞质注射法（59．20％）（P〈0．01）；成纤维细胞的重组成功率、卵裂率和囊胚率在两种方法间均无显著差异（P〉0．05）；在颗粒细胞，胞质注射法重组成功率（82．17％）显著高于电融合法（50．99％）（P〈0.05），其囊胚率也稍高于电融合法，但无显著差异（P〉0.05）；（3）上述3种供体细胞在冻融后支持重组胚发育力方面无显著差异（P〉0.05）。结论：在山羊体细胞核移植中，上述3种细胞支持重组胚的发育力以成纤维细胞和颗粒细胞较好；细胞冷冻对其无显著影响；在构建重组胚方法上，乳腺细胞以电融合法为宜，颗粒细胞是胞质注射法优于电融合法，而成纤维细胞两种方法均可。

The Study of Optimizing the Protocol of Somatic Nuclear Transfer on Goat

Nuclear transfer was carried out with mammary epithelial cells,fetal fibroblasts and cumulus-granulosa cells by method of intracytoplast injection and electro-fusion respectively.The influence of cell types,cell refrigeration and the methods of reconstruction of donor cells to developmental ability of cloned embryos were compared.The donor cells were treated with/without refrigeration,and were synchronized at G_0/G_1 stage by serum deprivation before nuclear transfer.Results:(1)The developmental ability of cloned embryos originated from mammary epithelial cells(blastocyst rate: 7.14%) was significantly lower than those from fetal fibroblasts(16.19%) and cumulus-granulosa cells(19.01%)(P<0.05).(2) There was no significant difference among three kinds of cell(P>0.05) mentioned above in the influence of cell refrigeration on developmental ability of cloned embryos.(3) The rate of reconstruction with electro-fusion(69.52%)(P<0.01)was significantly higher than that with intracytoplast injection(59.20%) to mammary epithelial cells.However,there was no significant difference between these two methods(P>0.05) for fetal fibroblasts.For cumulus-granulosa cells,the rate with intracytoplast injection(82.17%) was significantly higher than that with electro-fusion(50.99%)(P<0.05),and the rate of forming blastula was a little higher than that with electro-fusion,but not significantly different.Conclusions: Fetal fibroblasts and cumulus-granulosa cells were superior to sustain the developmental ability of cloned embryos.Cell refrigeration had no significant effect on the reconstruction of cloned embryos.Comparing the ways of reconstructing embryos,it was suggested that electro-fusion was better for mammary epithelial cells,but intracytoplasm injection was better than electro-fusion for cumulus-granulosa cells.Both methods were fit to fetal fibroblasts.