水牛体细胞核移植方法的研究

Studies on the Methods of Buffalo Somatic Cells Nuclear Transfer

探讨电融合参数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,在含有5 μg/mL细胞松弛素的操作液中进行去核,然后将经0.1 mg/L Aphidicolin (APD)处理1 d, 再用0.5% FBS培养2～9 d的水牛耳皮成纤维细胞或颗粒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5 μmol/L离子霉素激活处理5 min并在2 mmol/L的6-DMAP中培养3 h后,在含有颗粒细胞单层细胞的微滴中(30 μL)培养7～9 d,观察其卵裂和胚胎发育情况.当电融合的电场强度为1 500 V·μs/mm(电压100 V/mm×脉时15 μs)时,电脉冲3次,颗粒细胞核移植的融合率为74.18%,分裂率为71.82%,囊胚发育率为10%,融合率显著高于2次电脉冲(52.03%,P<0.05),卵裂率显著高于4次电脉冲(53.95%,P<0.05);当电脉冲次数为3次时,电场强度为2 000 V·μs/mm的分裂率(53.54%)显著低于1 500 V·μs/mm,2 500 V·μs/mm的融合率(62.0%)和分裂率(53.23%)均显著低于1 500 V·μs/mm(P<0.05).采用耳皮成纤维细胞作供核的融合率(59.75%)和分裂率(57.45%)均显著低于颗粒细胞.染色体组型分析显示,66.7%的核移植胚胎具有正常的供体细胞二倍体核型.将来自1头22岁的摩拉公牛耳皮成纤维细胞的2枚冻胚移植给受体母牛,妊娠到215 d时发生流产.结果表明,高龄水牛的体细胞核移植胚胎能发育到妊娠晚期;水牛体细胞核移植的适宜电融合参数为100 V/mm,15μs,3次电脉冲;颗粒细胞的电融合核移植效果优于耳皮成纤维细胞.

Effects of electrofusion parameters and donor cell types on the nuclear transfer of buffalo somatic cells 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 μg/mL cytochalasin B. Fibroblasts or granulosa cells cultured in DMEM supplemented with 0.1 μg/L Aphidicolin (APD) for 24 h and following in 0.5FBS for 2~9 days were transferred into enucleated oocytes by electronic fusion. The reconstructed embryos were activated by exposuring them to 5 μmol/L ionomycin for 5min and then culturing in 2 mmol/L 6-DMAP for 3h. After activation, embryos were cultured in 30 μL drop of granulosa cell monolayers for 7~9 days. When granulosa cells were used as donor cells and the direct current field intensity was fixed at 1 500 V·μs/mm applied, 3 pulses resulted in a significantly higher fusion rate (74.18) compared with 2 pulses (52.03) (P<0.05), and a significantly higher cleavage rate (71.82) in comparison with 4 pulses (53.95) (P<0.05). When the number of pulses was fixed at 3, increasing the direct current field intensity from 1 500 V·μs/mm to 2 000 or 2 500 V·μs/mm resulted in a significant decrease in either fusion rate or cleavage rate. The fusion rate (59.75) and cleavage rate (57.45) of fibroblast cells were significantly lower than that of granulosa cells. Karyotyping analysis showed that 66.7 of reconstructed embryos have normal diploid karyotype (48 XY), which was identical to that of donor cells. One recipient received 2 frozen/thawed embryos derived from the ear fibroblasts of a Mura bull aged at 22 years became pregnant and aborted at 215 days of gestation. These observations suggest that embryos derived from old buffalo somatic cells can develop to the late stage of gestation; the suitable electrofusion parameters on buffalo somatic cells nuclear transfer are 100 V/mm, 15 μs and 3 pulses; and granulosa cells are better than fibroblast cells used as donor karyoplasts for electrofusion in buffalo cloning.