水牛体细胞核移植相关影响因素的研究  

Studies on somatic cell nuclear transfer in buffalo

作　　者：陆凤花[1] 石德顺[1] 韦英明[1] 潘红平[1] 黄凤玲[1] 谭世俭[1] 王晓丽[1] 黄雅琼[1] 

机构地区：[1]广西大学动物繁殖研究所,广西南宁530005

出　　处：《广西农业生物科学》2008年第4期309-314,共6页Journal of Guangxi Agricultural and Biological Science

基　　金：国家"863"高科技研究发展计划项目(2002AA206651);国家自然科学基金(30460090);广西科学基金(桂科转0718005-3A和桂科青0640004);广西亚热带生物资源保护利用重点实验室开放课题(SB0602)

摘　　要：主要是探讨水牛卵母细胞的体外成熟与供体的年龄和性别对其核移植效果的影响。体外成熟培养22～24h的水牛卵母细胞，在含有5μg／mL细胞松弛素的操作液中进行去核，然后将经0．1μg／mL蚜栖菌素（Aphidicolin，APD）培养处理24h，再用0．5％胎牛血清（FBS）培养2d的水牛耳皮成纤维细胞注射到去核的卵母细胞卵周隙中，再经电融合形成重构胚。重构胚经5μmol／L离子霉素激活处理5min并在2mmol／L的二甲氨基嘌呤（6-DMAP）中培养3h后，在含有颗粒细胞单层细胞的微滴中（30μL）培养7d，观察其卵裂和胚胎发育情况。结果显示：在添加有10ng／mL上皮生长因子（EGF）成熟液中培养的受体水牛卵母细胞的融合率和重组胚卵裂率与对照组无显著差异（P〉0．05），但其囊胚发育率明显高于对照组（18．53％vs．7．56％，P〈0．05）。来自胎儿（3个月）或成年（一头5～6岁，另一头22岁）水牛的耳皮成纤维细胞核移植后重组胚的卵裂率差异不显著（P〉0．05），但水牛胎儿体细胞构建的重组胚囊胚率显著高于成年体细胞（22．55％vs．10．77％和8．09％，P〈0．05）。雌性水牛成纤维细胞构建的重组胚的囊胚发育率（20．23％）显著高于雄性水牛体细胞的囊胚率（10．16％，P〈0．05）。以上结果表明：（1）成熟液中添加EGF能提高受体卵母细胞核移植后的胚胎发育率；（2）水牛体细胞核移植效果受供体的个体年龄和性别的影响：胎儿成纤维细胞的核移植效果优于成年的成纤维细胞，而且以雌性的效果较好。Development of nuclear transfer （NT） embryos reconstructed with different source of buffalo recipient oocytes and donor cells were investigated in the present study. Buffalo oocytes obtained from ovaries at slaughter were matured in vitro for 22 -24 h and then enucleated in manipulation medium containing 5 μg/mL cytochalasin B. Buffalo fibroblasts cultured in Dulbecco＇s Modified Eagle Medium （DMEM） supplemented with 0. 1 μg/mL Aphidicolin （APD） ＋0. 5% fetal bovine serum （FBS） for 24 h and following in 0.5%FBS for 2 days were transferred into enucleated oocytes by electronic fusion. The reconstructed embryos were activated by exposuring them to 5 μmol/L ionomycin for 5 min and then culturing in 2 mmol/L 6-DMAP for 3 h. After activation.embryos were cultured in 30 μL drop of granulosa cell monolayers for 7 days. More NT embryos derived from oocytes matured in the maturation medium containing 10 ng/mL epithelial growth factor （EGF） developed to blastocysts compared with that of embryos developed from oocytes matured in the maturation medium without EGF （P 〈 0. 05）. The blastocyst developmental rate of embryos derived from fetal fibroblasts was significantly higher than that of embryos derived from adult fibroblasts, moreover, the percentage of blastocysts development of embryos reconstructed with female fibroblasts was also significantly higher than that of embryos reconstructed with male fibroblasts. In conclusions, EGF can improve the developmental competence of buffalo NT embryos. Fibroblasts derived from different age and sex of donor have a profound effect on the development of buffalo NT embryos.

关 键 词：水牛 核移植 成纤维细胞 卵母细胞 胚胎 

分 类 号：S814.8[农业科学—畜牧学] S823[农业科学—畜牧兽医]