两种不同玻璃化冷冻方案对小鼠卵母细胞纺锤体的影响  

Influence of Two Different Vitrification Cryopreservation Methods on Spindles of Mouse Oocytes

作　　者：孙海翔[1] 胡娅莉[1] 陈林君[1] 张宁媛[1] 徐志鹏[1] 

机构地区：[1]南京大学医学院附属鼓楼医院生殖医学中心,江苏南京210008

出　　处：《中华男科学杂志》2006年第12期1076-1079,1083,共5页National Journal of Andrology

基　　金：江苏省自然科学基金(BK2004095)

摘　　要：目的:探讨两种不同的玻璃化冷冻方案对小鼠第二次减数分裂中期卵母细胞纺锤体的影响。方法:实验分3组:方案A组、方案B组和对照组(新鲜卵母细胞)。均以冷冻环为载体,方案A组用乙二醇(EG)作为冷冻保护剂,方案B组用乙二醇联合二甲亚砜(DMSO)作为冷冻保护剂,用直接投液氮高速制冷冷冻小鼠卵母细胞;解冻后3h小鼠卵母细胞经固定并用间接免疫荧光法染色微管及染色体,观察纺锤体及染色体的形态。结果:两种玻璃化冷冻方案中,卵母细胞解冻后的存活率差异无显著性(80.3%vs87.5%,P>0.05);经玻璃化冷冻方案A冷冻的卵母细胞解冻后具有完整纺锤体结构的卵母细胞数显著低于对照组和方案B组(15.2%vs78.7%,15.2%vs77.5%,P<0.05),后两者差异无显著性(78.7%vs77.5%,P>0.05);方案A组解冻后具有正常染色体的卵母细胞数显著低于对照组和方案B组(17.4%vs76.6%,17.4%vs72.5%,P<0.05),后两者差异无显著性(76.6%vs72.5%,P>0.05);方案A组异常染色体数显著高于对照组和方案B组(82.6%vs19.1%,82.6%vs27.5%,P<0.05),后两者差异无显著性(19.1%vs27.5%,P>0.05)。结论:改变玻璃化冷冻方案有利于保持解冻后小鼠卵母细胞纺锤体结构完整性。Objective ： To investigate the influence of two different vitrofication oocytes. Methods： Three groups were included in the experiment, Group A, cryopreservation methods on the spindles of mouse M Group B and the control（ fresh oocytes）. Mouse oocytes were vitrified by using cryoloop, with ethylene glycol（EG） in Group A and with EG ＋ dimethyl sulphoxide（DMSO） in Group B as cryoprotectants, and then the oocytes were placed directly into liquid nitrogen. Three hours after the frozen oocytes were thawed they were fixed, and the microtubulin and chromosome were stained by indirect immunofluorescent method. Results： The survival rates of the oocytes after treated by the two vitrification cryopreservation methods had no difference（ 80.3% vs 87.5% , P 〉 0.05 ）. The rate of the intact spindles in Group A was much lower than that of the control and Group B（ 15.2% vs 78.7% , 15.2% vs 77.5% , P 〈 0.05 ）. But there was no difference between the latter two groups（78.7% vs 77.5% , P 〉 0.05 ）. The oocytes with normal chromo- some in Group A were much less than in the control and Group B （ 17.4% vs 76.6% , 17.4% vs 72.5% , P 〈 0.05 ）, with no difference between the latter two groups （76.6% vs 72.5% , P 〉 0.05 ） ; The oocytes with abnormal chromosome were more in Group A than in the control and Group B （82.6% vs 19.1%, 82.6% vs 27.5%, P 〈0.05）, with no difference between the latter two groups（19.1% vs 27.5% , P 〉 0.05）. Conclusion： The changed vitrification cryopreservation method helps conserve the intact spindleconfiguration of mouse oocytes.

关 键 词：卵母细胞 纺锤体 玻璃化冷冻 小鼠 

分 类 号：R318.52[医药卫生—生物医学工程]