兔着床后全胚胎培养方法的建立

The establishment of postimplantation rabbit whole embryo culture

目的:建立用于发育毒物研究和评价的兔着床后全胚胎培养方法。方法:取怀孕第9天(gestation day 9,GD9)的孕兔胚胎,参照Carney和Naya的方法,剥离胚胎后单瓶培养,体外培养48 h后将发育情况与文献报道的结果进行对比;并用5.0mmol/L的甲氧基乙酸(methoxyacetic acid,MAA)作为致畸阳性物对所建立的培养方法作进一步验证,采用Carney建立的兔胚胎形态评分系统进行评分。结果:本研究中23个GD9兔胚胎体外培养48 h后,存活率为100%,头长平均增长2.15倍,卵黄囊闭合和胚胎弯曲情况、体节数、自发畸形率、形态评分均数等指标均与文献报道的体外培养发育情况相近,5.0 mmol/L的MAA显示对兔胚胎有发育毒性,包括卵黄囊闭合减少、头部肿胀、面部器官缺失、体节紊乱、尾神经管闭合不全等,形态评分低于溶剂对照(P<0.05)。结论:本研究所建立的兔着床后全胚胎培养方法是可行的。

OBJECTIVE：To establish post-implantation rabbit whole embryo culture for studying teratogenic agents.METHODS：Gestation day 9（GD9） rabbit embryos were cultured mainly according to the method of Carney and Naya,embryos dissected,and cultured one per bottle,and morphology was compared with results in the literature after culturing 48 h in vitro.5 mmol/L methoxyacetic acid was used as positive control.The scoring system was a new refined morphological scoring system by Carney.RESULTS：23 GD9 embryos cultured in vitro all survived,the average growth of head length was 2.15 times,the visceral yolk sac closure,flexion,somites,spontaneous malformation,average of morphology scores etc were close to the literature.The positive control of 5 mmol /L MAA induced developmental toxicity consisted of reducing closure rate of yolk sac,swollen head regions,lack of facial development,disordered somites,delayed closure of caudual neural tube etc,the scoring was lower than the solvent control（P0.05）. CONCLUSION：The post-implantation rabbit whole embryo culture method and conditions established in our laboratory was reliable,which could be an efficient tool to study teratogenic agents.