水牛成纤维细胞核移植的研究

Development of Buffalo Embryos Reconstructed by Nuclear Transfer of Adult Fibroblasts

本研究对影响水牛耳部成纤维细胞核移植效果的因素进行了探讨。体外成熟 2 2h的水牛卵母细胞经显微操作去核后 ,将一个经传代培养的水牛耳部成纤维细胞注入到胞质内。经核移植的水牛卵母细胞用 5μM离子霉素激活处理 5min ,然后在含有 2mM 6 -甲二氨基嘌呤 ( 6 -DMAP)的培养液培养 3h。当成纤维细胞用DNA合成抑制剂Aphidicolin( 0 .4μg/ml)培养 1d或 2d再进行核移植时 ,其囊胚发育率 ( 2 .0 %和 0 )明显低于 ( p <0 .0 5)对照组 ( 4.9% ) ,虽然其分裂率 ( 6 0 .9%和 6 0 .5% )与对照组 ( 6 2 .9% )无明显差异 ( p >0 .0 5)。当成纤维细胞的培养代数由G5增加到G6和G7时 ,囊胚发育率亦随之下降 ( 5.3% ,2 .0 %和 1 .8% ,p <0 .0 5) ,但分裂率无显著差异 ( 6 8.0 % ,6 0 .9%和 6 0 .7% ,p >0 .0 5)。结果表明 ,在现有核移植实验条件下 ,水牛成纤维细胞的培养代数应控制在 5代之内 。

Factors affecting the in vitro development of buffalo embryos reconstructed by nuclear transfer of adult ear fibroblasts were investigated in this study. Buffalo oocytes matured in vitro for 22 h were enucleated by micromanipulation and an in vitro cultured ear fibroblast was introduced into the cytoplasts by microinjection. Reconstructed oocytes were activated at 26-28 h by exposure them to 5 μm ionomycin for 5 min and 2 mM 6-dimethyl-aminopurine for 3 h at 38.5℃ and 5% CO 2 in air. The blastocyst development of nuclear transfer embryos decreased when ear fibroblasts were pretreated with 0.4 μg/ml aphidicolin for 1 day (2.0%) or 2 days (0%), in comparison with ear fibroblasts cultured with DMEM supplemented with 10% FCS (4.9%), although the cleavage rate was similar among the three groups (60.9%, 60.5% and 62.9% respectively). The proportion of reconstructed oocytes developing into blastocysts was also trended to decrease as the ear fibroblast culture passages progressed from G5 (5.3%) to G6 (2.0) and G7 (1.8%), although there was no significant difference among the three groups (68.0, 60.9 and 60.7% respectively). Results indicate that aphidicolin is harmful to buffalo ear fibroblast nucleus reprogram, buffalo ear fibroblast culture passages should be controlled within 5 generations under the present conditions.