兔卵巢组织慢速冷冻和玻璃化冷冻保存方法的研究

Study of slow-cooling and vitrification for rabbit ovarian tissue cryopreservation

目的比较不同冷冻方法对兔卵巢组织的保存效果。方法 15只新西兰雌兔,取卵巢皮质切块后,随机分成5组:新鲜组(A组)、二甲亚砜慢速冷冻组(B组)、丙二醇慢速冷冻组(C组)、冷冻管玻璃化冷冻组(D组)及固相表面玻璃化冷冻组(E组)。比较各组卵巢组织的原始和初级卵泡形态正常率;通过体外培养测定培养液中雌二醇(E_2)水平;比较冻融后卵巢组织的内分泌功能;通过TdT介导的dUTP缺口末端标记技术,检测细胞凋亡情况。结果原始卵泡正常形态率与A组相比,B、D组显著降低(P<0.05),C、E组无显著性差异;初级卵泡正常形态率5组间无显著性差异。培养14 d后,各实验组原始卵泡形态正常率均低于A组(P<0.001),各实验组间无显著性差异;组间初级卵泡形态正常率无明显差异。体外培养过程中,培养液E_2水平不断增加(P<0.001),慢速冷冻和玻璃化冷冻的E_2平均浓度有显著性差异(P<0.05)。细胞凋亡检测显示,无论是原始卵泡还是初级卵泡,与A组相比,各实验组的卵泡凋亡率均无显著性差异。结论本实验中,慢速冷冻和固相表面玻璃化冷冻均能很好地保存兔卵巢组织,经过体外培养,能保持一定的内分泌功能。

Objective： To compare the protective effect on rabbit ovarian tissue with different cryopreservation methods. Methods： The ovarian biopsies from 15 New Zealand female rabbits were randomly divided into fresh （Group A）, Dimethyl sulfoxide （DMSO） slow-cooling（Group B）, propylene glycol （PROH） slow-cooling （Group C）, tube vitrification （Group D）, solid-surface vitrification （Group E） groups. The rates of morphological integral follicles were compared between fresh and the post-freezing/thawing tissue. The levels of estradiol in culture medium were measured to compare the endocrine functions of the post- cryopreservation tissue. The apoptotic follicles in ovarian tissue were detected by TdT-mediated dUTP nick end labeling. Results： Compared with Group A, the percentages of morphologically normal primordial follicles significantly reduced （P〈0.05） in Group B and D, and there were no significant differences in Group C and E. The percentages of morphologically normal primary follicles were not different among 5 groups. After 14 days in-vitro culture, all cryopreservation groups showed significant reduction of the proportions of primordial follicles in cultured tissues compared with Group A （P〈0. 001）. The proportions of primary follicles were not different among 5 groups. The levels of estradiol in culture medium gradually increased during the 14 days in-vitro culture （P〈0. 001）. The difference of estradiol levels between slow-cooling and vitrification were significant （P=0. 021）. The percentages of apoptotic follicles were not different among all groups. Conclusion： Both slow-cooling and vitrification for rabbit ovarian tissue cryopreservation were effective, and the endocrine functions of the post-cryopreservation tissue could be preserved satisfactorily.