激活方法对大鼠-小鼠种间体细胞核移植重组胚发育的影响

目的：探讨电激活及化学激活对大鼠-小鼠种间体细胞核移植重组胚发育的影响。方法：采用血清饥饿法同步化处理的SD大鼠成纤维细胞作为供体细胞，常规超排KM小鼠获得卵母细胞，采用盲吸法去核，显微直接注核法构建重组胚，分别采用6-DMAP 乙醇、放线茵酮、透明质酸酶3种化学激活方法和不同电场强度、脉冲电激活重组胚，CZB培养液中培养，观察重组胚发育。结果：电激活：电场强度(V/cm)，脉冲时间(ms)为：1000/100，1500/30，1800/30，2400／40时，卵裂率分别为：17．39％，28．07％，26．52％，0．00％。化学激活：使用透明质酸酶、放线茵酮、6-DMAP 乙醇时卵裂率分别为：5．56％，25．00％和33．91％。结论：电激活和化学激活均可有效激活重组胚：电激活时当电压为1500～1800V/cm、脉冲为30～40ms时，有利于重组胚的发育，电压过高时会将重组胚击碎；本实验所有激活方法中，6-DMAP 乙醇激活法所得卵裂率和桑椹胚率最高。

Effect of electrical and chemical activation on reconstructed embryo formed by rat-mouse inter-species somatic cell nuclear transplantation

Objective:To study the effect of electrical and chemical activation on reconstructed embryos formed by rat-mouse inter-species somatic cell nuclear transplantation.Methods:Cultured SD rat fibroblast cells for donor of nuclear transfer were treated with serum starvation. The donor nuclei were injected into the enucleated oocytes that were super discharged from KM mice. Then these reconstructed embryos were activated by electrical activation at different electrical field strength and impulse and chemical activation with 6-DMAP+ethanol, cycloheximide and hyaluronidase, and cultured in CZB medium.Results:In electrical activation, when electrical field strength and pulse were 1?000?V/cm and 100?ms,1?500?V/cm and 30?ms,1?800?V/cm and 30?ms,2?400?V/cm and 40?ms, the cleavage rates were 17.39%, 28.07%,26.52% and 0.00%,respectively. In chemical activation, the reconstructed embryos were activated by hyaluronidase, cycloheximide and 6-DMAP+ethanol and the cleavage rates were 5.56%, 25.00% and 33.91%,respectively.Conclusion:Electrical and chemical methods can effectively activate reconstructed embryo. The condition of electrical field strength from 1?500?V/cm to 1?800?V/cm and pulse from 30?ms to 40ms are the best way to activate reconstructed embryo formed by rat-mouse inter-species somatic cell nuclear transplantation. The rates of cleavage and morula activated by 6-DMAP+ethanol were the highest among all metheds used.