猪体细胞核移植电融合参数的研究

Studies on the electric fusion parameters for nuclear transfer of porcine somatic cells

本研究用体外成熟42-46h的猪卵母细胞为核移植受体，猪颗粒细胞为核移植供体，对猪体细胞核移植的电融合参数进行了探讨。结果发现，当脉冲时间为40μs时，0．77kV／cm组的融合率（59．3％）明显高于1．54kV／cm组（38．6％，P〈0．05），分裂率（77．8%）及桑椹胚／囊胚发育率（33．3％）显著高于2．31kV／cm组（52．2％，13．6％，P〈0．05）；当电场强度为0．77kV／cm时，20μs组的融合率（68．0％）明显高于30μs组和40μs组（42．7％和35．5％，P〈0．05），分裂率（89．3％）明显高于脉冲时间为40μs组（60．5％）；当电场强度为0．77kV／cm，脉冲时间为20μs时，电脉冲1次的融合率（72．8％）明显高于电脉冲2次（56．0％，P〈0．05），在直流电脉冲前是否施加交流电（1．0V）对融合率（70．0％vs76．0％）、分裂率（77．3％vs75．0％）以及胚胎发育率（17．3％vs18．4％）均无显著影响（P〉0.05）。以上结果表明，在本所实验室条件下，猪体细胞核移植中的适宜电融合参数为：0．77kV／cm，20μs，电脉冲1次，这将为随后的猪体细胞核移植奠定了基础。

The appropriate electric fusion parameters for nuclear transfer （NT） of porcine somatic cells were investigated using in vitro matured porcine oosytes as recipients and granulose cells as donors in this study. When the pulse duration was fixed at 40 μs, 0. 77 kV/cm of field intensity resulted in significantly more oocytes fused （59. 3%） in comparison with 1.54 kV/cm （38. 6%, P〈0.05）, and more NT embryos cleaved （77.8%） and developed to the morula and blastocyst stage （33.3%） in comparison with 2.31 kV/cm （52.2% and 13.6% respectively, P〈0.05）. When the field intensity was fixed at 0. 74 kV/cm, 20μs of pulse duration resulted in more oocytes fused （68.0%） in comparison with 30 μs （42.7%） and 40 μs （35.5%, P〈0.05）, and more oocytes cleaved （89. 3%） compared with 40 μs （60. 5%, P〈0. 05）. When the field intensity was fixed at 0.74 kV/cm and pulse duration was fixed at 20 μs, one pulse resulted in more oocytes fused （72.8%） in comparison with two pulses （56.0%, P〈0. 05）. Applying alternating current （1.0 V）before direct pulsedid not affect the fusion rate （70.0% vs 76.0%）, cleavage rate （77.3% vs 75.0%） embryo development （17. 3% vs 18.4%, P〉0. 05）.In conclusion, the appropriate electric parameter for of porcine somatic NT is proved to be 0. 77 kV/cm of field intensity, 20 μs of pulse duration and 1 direct pulse, which will benefit the porcine somatic NT in future.