CAJ | 학술논문

目的探寻一种稳定有效的卵母细胞激活方法及体外培养体系,为下一步的治疗性克隆胚激活及培养实验提供相关参照. 方法 (1)选取具有第一极体的成熟兔卵母细胞随机分组,比较1.2 kV/cm、1.6 kV/cm、2.0 kV/cm场强的电激活及5μg/mL离子霉素(ION)激活对兔卵母细胞孤雌激活效果；(2)激活处理后的卵母细胞,分别在添加0、10、20、40、80 ng/mL白血病抑制因子(LIF)及添加0、1×、2×胰岛素、转铁蛋白、硒化钠复合物(ITS)的M199液中培养,比较孤雌胚胎的卵裂率、8-16细胞胚胎比例、桑囊率以及囊胚细胞总数情况. 结果 (1)不同激活方式对兔卵母细胞的激活效果差异较大.ION处理组存活率高于2.0 kV/cm电激活处理组,差异有统计学意义(P＜0.05)；而在分裂率、8-16细胞率、桑囊率上,ION处理组高于1.2 kV/cm电激活处理组,差异有统计学意义(P＜0.05).(2) 20 ng/mL LIF组桑囊率高于80 ng/mL LIF组,差异有统计学意义(P＜0.05).在囊胚细胞总数上,20 ng/mL LIF组高于40 ng/mL LIF和80 ng/mL LIF组,差异有统计学意义(P＜0.05).1×ITS组的桑囊率高于2×ITS组,差异有统计学意义(P＜0.05).此外,0×ITS组及1×ITS组的囊胚细胞总数分别为166.00枚、147.40枚,多于2×ITS组(78.00枚),差异有统计学意义(P＜0.05). 结论 ION处理激活新西兰大白兔卵母细胞能获得较好的分裂率和囊胚率；添加20 ng/mL LIF、1 ×ITS可以促进兔孤雌激活胚胎的后期发育.
목적 탐심일충은정유효적란모세포격활방법급체외배양체계,위하일보적치료성극륭배격활급배양실험제공상관삼조. 방법 (1)선취구유제일겁체적성숙토란모세포수궤분조,비교1.2 kV/cm、1.6 kV/cm、2.0 kV/cm장강적전격활급5μg/mL리자매소(ION)격활대토란모세포고자격활효과；(2)격활처리후적란모세포,분별재첨가0、10、20、40、80 ng/mL백혈병억제인자(LIF)급첨가0、1×、2×이도소、전철단백、서화납복합물(ITS)적M199액중배양,비교고자배태적란렬솔、8-16세포배태비례、상낭솔이급낭배세포총수정황. 결과 (1)불동격활방식대토란모세포적격활효과차이교대.ION처리조존활솔고우2.0 kV/cm전격활처리조,차이유통계학의의(P＜0.05)；이재분렬솔、8-16세포솔、상낭솔상,ION처리조고우1.2 kV/cm전격활처리조,차이유통계학의의(P＜0.05).(2) 20 ng/mL LIF조상낭솔고우80 ng/mL LIF조,차이유통계학의의(P＜0.05).재낭배세포총수상,20 ng/mL LIF조고우40 ng/mL LIF화80 ng/mL LIF조,차이유통계학의의(P＜0.05).1×ITS조적상낭솔고우2×ITS조,차이유통계학의의(P＜0.05).차외,0×ITS조급1×ITS조적낭배세포총수분별위166.00매、147.40매,다우2×ITS조(78.00매),차이유통계학의의(P＜0.05). 결론 ION처리격활신서란대백토란모세포능획득교호적분렬솔화낭배솔；첨가20 ng/mL LIF、1 ×ITS가이촉진토고자격활배태적후기발육.
Objective To investigate an appropriate activation method and culture system in vitro of rabbit parthenogenetic embryos to make an experimental foundation for therapeutic cloning.Methods Mature rabbit oocytes with polar body 1 (PB 1) were divided randomly into four groups and accepted,respectively,parthenogenetic activation with 1.2 kV/cm,1.6 kV/cm and 2.0 kV/cm electricity stimulation or 5 μg/mL ionomycin (ION) chemical treatment; the effects of these different treatments on activation efficiency were compared.The activated oocytes were cultured,respectively,in M199 supplemented with various concentrations of leukaemia inhibitory factor (LIF,0,10,20,40 and 80 ng/mL) or insulin+transferrin+sodium selenite (ITS,0×,1 × and 2×); and then,the rate of cleaved oocytes,percentage of morulas/blastocysts and total number of blastocysts were compared between each two groups.Results (1) Different treatments on the oocytes showed significantly different activation effects.The survival rate of oocytes activated in 5 μg/mL ION for 4 min were significantly higher than that of ones stimulated by 2.0 kV/cm electricity treatment (P=0.016).The percentages of cleaved oocytes,developing 8-16 cells and morulas/blastocysts in the ION treatment group were also significantly higher as compared with those in the 1.2 kV/cm electric treatment group (P=0.000,P=0.002 andP=0.026,respectively).(2) Significant difference in the percentage of morulas/blastocysts was noted between 20 ng/mL and 80 ng/mL of LIF supplementation (P=0.003); in addition,the total number ofblastocysts in the medium supplemented with 20 ng/mL LIF was significantly different to those with 40 ng/mL and 80 ng/mL,respectively (P=0.011,P=0.002); In experiment of ITS supplementation,significant difference in the percentage ofmorulas/blastocysts was shown between 1 × and 2× ITS supplementation (P=0.003); in addition,the total number of blastocysts in the medium supplemented with 0× or 1 × ITS (166 and 147 cells) was significantly different as compared with that with 2× ITS (78 cells,P=0.015,P=0.044).Conclusion The parthenogenesis activation of the New Zealand rabbit's oocytes treated with ION+6-DMAP shows a higher rate of cleaved oocytes and blastocysts than that with electricity stimulation; the subsequent development of parthenogenetic embryos in vitro could be significantly improved by supplementing with 20 ng/mL LIF and 1× ITS.