小鼠卵母细胞电激活的实验研究

Experimental Study on Electrical Activation of Oocytes of Mouse

目的寻找用电激活方法作小鼠体细胞克隆的适宜卵龄的卵母细胞及电激活参数。方法在0.6kV/cm、80μs、2个电脉冲(2p)下,比较了注射人绒毛膜促性腺激素(HCG)后13、16、19、22h卵母细胞的激活率、碎裂+死亡率。在上述相同电脉冲下,统计了经体外培养(IVC)和新鲜的卵龄为16、19、22h的卵母细胞的激活率,碎裂+死亡率;比较了场强分别为0.6、0.9、1.2、1.5、1.8kV/cm,电压时程分别为10、20、40、80、100、150、200μs,2p对注射HCG后16h卵母细胞的电激活效果。结果注射HCG后16、19h卵母细胞电激活率较高,分别为63.6%和76.7%,经体外培养后激活率下降,碎裂+死亡率明显增加。在0.9kV/cm、80/100μs,2p时激活率达70%～80%。在1.2kV/cm、40/80/100μs、2p时,激活率达到了80%左右,碎裂+死亡率低于16%。结论注射HCG后16h的卵母细胞为适宜的小鼠核移植的受体细胞。0.9kV/cm、80/100μs,2p及1.2kV/cm、40/80/100μs、2p为适宜的电激活参数。

Objective To find out the suitable age of oocyte and parameters of electrical activation in the process of mouse somatic cloning. Methods The rate of the activated egg and the rate of lysed＋dead egg were compared at different ages of oocytes： 13, 16, 19, 22 h （after HCG injection） with the electrical parameters of 0.6 kV/cm, 80 us, 2 electrical pulses （2p）. The effects of electrical activation bn the in vitro cultured and fresh egg at the same ages： 16, 19, 22 h （after HCG injection） were also compared. The activation effects of oocytes 16 h after HCG injection were observed under different voltage strengths： 0.6, 0.9, 1.2, 1.5 and 1.8 kV/cm and different voltage durations： 10, 20, 40, 80, 100, 150 and 200us. Results The activation rate of 16-h and 19-h oocytes was high up to 63.6 % and 76.7 %0 respectively, but it was decreased and the lysed＋dead rate increased obviously after in vitro culture, especially of 19-h oocytes. When the parameters were 0.9 kV/cm, 80/100us, 2 p and 1.2 kV/cm, 40/80/100us, 2 p, the activation rate was up to 70 %-80% and the lysed＋dead rate was less than 16 %. Conclusion The oocytes 16 h after HCG injection was the ideal recipient cell used for somatic cloning. 0. 9 kV/cm, 80/100 us, 2 p and 1.2 kV/cm, 40/80/100us, 2 p were suitable parameters of electrical activation.