【摘要】 以水牛耳皮成纤维细胞为供体细胞,采用电融合方法,探讨细胞松弛素B(CB)对水牛体细胞核移植效果的影响。体外成熟培养22~24 h的水牛卵母细胞去核后,将经0.1 mg/L Aphidicolin(APD)+0.5%FBS培养2~9 d的水牛耳皮成纤维细胞注射到卵周隙中再经电融合(100 V/mm,15μs,电脉冲3次)构建核移植重构胚。重构胚经化学激活后(5μmol/L)离子霉素5 min,2 mmol/L 6-DMAP 3 h)培养,7~9 d评定其胚胎发育能力。结果显示,在含CB(3 mg/L)的融合液中进行电融合后,核移植的融合率、重组胚的存活率、卵裂率和囊胚率与对照组(不含CB)相比均无显著差异(P>0.05);核移植重组胚激活前用含CB(6 mg/L)的培养液培养1 h,其激活后的存活率(97.52%)和体外囊胚发育率(22.09%)均显著地高于未经CB处理的重组胚的存活率(93.87%)和囊胚率(13.25%,P<0.05);重组胚经离子霉素激活5 min后,在6-DMAP+CB中培养3 h的分裂率明显低于放在6-DMAP中培养3 h的分裂率(65.37%vs 78.92%,P<0.05),但囊胚发育率无显著差异(11.19%vs 10.96%,P>0.05)。这表明水牛体细胞核移植电融合时,融合液中不添加CB,而核移植重组胚激活前经CB培养处理后,有利于胚胎的进一步发育,但激活后用CB培养处理会降低胚胎的发育率。更多还原

【Abstract】 Effect of the cytochalasin B(CB) on the development of buffalo somatic cell nuclear transfer(NT) embryos were investigated in this study.Buffalo oocytes obtained from ovaries at slaughter were matured in vitro for 22-24 h and then enucleated in manipulation medium containing 5 mg/L cytochalasin B.Fibroblasts in DMEM+10%FBS were treated with 0.1 mg/L aphidicolin(APD) for 1 day and 0.5%FBS for 2-9 days were transferred into enucleated oocytes by electronic fusion(100 V/mm,15 μs and 3 pulses).The reconstructed embryos were activated by exposuring them to 5 μmol/L ionomycin for 5 min and then culturing in 2 mM 6-DMAP for 3 h.After activation,embryos were cultured in 30 μL drop of granulosa cell monolayers for 7-9 days,to evaluate the embryonic development.Addition of CB 3 mg/L to electrofusion manipulation medium,there was no significant difference in the fusion rate,survival rate,cleavage rate and blastocyst development of the nuclear transfer embryos as compared with the control group(P>0.05).Treatment of reconstructed embryos with 6 mg/L CB for 1 h before activation resulted in more embryos survived and developed to blastocysts,but there was a significant decrease in the cleavage rate when treated them with 6 mg/L CB for 3 h after activation.In conclusions,addition of CB to electrofusion medium has no profound effect on the development of buffalo NT embryos and there was Not a beneficial effect on their development when treated buffalo NT embryos with CB after activation,but treated them with CB before activation can improve their development.更多还原