小鼠卵巢组织冷冻保存方法对组织内分泌功能的影响

Impact of different cryopreservation method of mice ovarian tissue on their endocrine function

目的比较不同的冷冻方法对小鼠卵巢组织的保存效果及对组织内分泌功能的影响。方法 25只6周龄ICR雌鼠,取卵巢皮质切块后,根据不同冷冻方法随机分成5组:新鲜对照组、二甲亚砜慢速冷冻组(A组)、丙二醇慢速冷冻组(B组)、冷冻管玻璃化冷冻5min组(C组)及冷冻管玻璃化冷冻8min组(D组)。分别进行卵巢组织冷冻复苏,复苏后体外培养2周以及复苏后同种移植饲养2周实验。比较各组体外培养液中以及复苏移植后小鼠静脉血中雌二醇(E2)水平。结果体外培养过程中,各组培养液中E2水平均随培养时间不断增加(F=14.146,P<0.001);同一天的E2水平比较,A、B组显著高于对照组及C、D组(P<0.05),C、D组与对照组无统计学差异(P>0.05)。移植饲养2周后,DMSO慢速冷冻复苏移植组、PROH慢速冷冻复苏移植组的平均E2水平显著高于两组玻璃化复苏移植组、新鲜组织移植对照组、未处理自然对照组及去势未移植对照组(P<0.05),新鲜移植对照组、两组玻璃化复苏移植组及去势未移植对照组的平均E2水平显著低于未处理的自然对照组(P<0.05)。结论慢速冷冻及玻璃化冷冻的小鼠卵巢组织复苏后,经过体外培养及同种移植均能保持一定的内分泌功能;慢速冷冻效果可能优于冷冻管玻璃化冷冻。

Objective：To study the effect of slow-freezing or vitrification for cryopreservation of mouse ovarian tissue on their endocrine function.Methods：The ovaries of 25 ICR mice were cut into pieces of 1mm×1mm×1mm after the vessels and other connective tissues were removed.Then the ovarian tissues were randomly divided to five groups,i.e.fresh tissue（control group）,dimethyl sulfoxide slow-freezing group（group A）,propylene glycol slowfreezing group（group B）,tube vitrification for 5min group（group C）and tube vitrification for 8min group（group D）,with 5ovaries for each group.The frozen/thawed tissues were cultured in vitro for two weeks,and parts of the tissues were transplanted to the castrated ICR mice under the kidney capsule.The estradiol（E2）levels in the collected culture medium and in serum were measured and compared among the five groups.Results：When the ovarian tissues were cultured in vitro,E2 levels were increased consistently in all of these groups（P〈0.001）during the 14 days of culture.The E2 levels were higher in group A and B than those in group C,D and control group on every specific day（P0.05）.In the transplanted groups,E2 levels were higher in group A and B than in group C,D and control group on day 14 after transplanted （P〈0.05）.Conclusions：Both of slow-freezing and vitrification can preserve ovarian endocrine function in mouse.The slow-freezing method is better than tube vitrification method.