摘要
目的:探讨钙离子载体A23187或联合嘌呤霉素(puromycin)对人类体外成熟卵母细胞的孤雌激活作用。方法:收集体外培养成熟的人卵母细胞124枚,根据体外成熟培养的时间分为24、48、72h组。分别采用A23187、A23187联合嘌呤霉素进行孤雌激活,然后应用荧光原位杂交(FISH)技术对孤雌胚胎进行性染色体分析。结果:A23187或联合嘌呤霉素能激活体外成熟的卵母细胞,激活率分别为38.9%、71.8%;二者联合应用能有效地提高孤雌胚胎的发育潜能。随着体外成熟培养的时间延长,A23187联合嘌呤霉素对卵母细胞激活率呈下降趋势;其中24、48h组成熟的卵母细胞激活率和胚胎发育潜能显高于72h组。FISH对9个孤雌胚胎的性染色体分析示,7个孤雌胚胎为XX,2个为X。结论:钙离子载体A23187联合嘌呤霉素能有效地激活人体外成熟卵母细胞,形成的孤雌胚胎核型多数为双倍体。
Objective： To investigate the parthenogenetic activation of calcium ionophore A23187 and puromyein on the human oocytes matured in vitro. Methods： Immature oocytes at germinal vesicle stage or metaphase of first meiosis from immature oocyte aspiration were harvested and cultured in vitro. Following in vitro maturation, 124 mature oocytes were derided into 3 groups according to the time of maturation in vitro： 24-hour group （n=18）, 48-hour group （n=84）, 72-hour group （n=22）. The oocytes were exposed to A23187 （5μmol/L） for 5 minutes or subsequently were treated with puromycin （10μg/ml） for 4 hours. The activation rate, proportion of oocytes that showed pronucleus formation and second polar body extrusion were calculated at 16～18 hours after activation. Sex chromosomal analysis of the parthenogenetic embryos was performed by dual color fluorescence in situ hybridization. Results： The activation rate in 48-hour group that the oocytes showed at least one pronucleus （PN） after treatment were 38.9% （7/18） in the A23187 group and 71.8% （28/ 39） in the combination of A23187 and puromycin. Cleavage rate and the developmental potential of parthenogenones in combinative group were significantly higher than in the A23187 and control groups, re- spectively. Furthermore, the activation rate in 24-hour group was similar to the 48-hour group＇s, which was significantly higher than 72-hour group＇s. FISH analysis showed 7 embryos with XX and two embryos with X in 9 parthenogenetic embryos. Conclusion： The combination of A23187 with puromycin proved to be an effective method of producing diploid parthenogenones.
出处
《山东大学学报（医学版）》
CAS
北大核心
2005年第8期685-689,共5页
Journal of Shandong University:Health Sciences
基金
国家自然科学基金资助课题(39300136)。
关键词
卵母细胞
离子栽体
嘌呤霉素
孤雌激活
荧光原位杂交
Oocytes
Ionophores
Puromycin
Parthenogenetic activation
Fluorescence in situ hybridization
