成年小鼠雄性生殖细胞的冷冻保存  

Cryopreservation of Adult Mouse Testicular Germ Cells

作　　者：李莲军[1] 阮承迈[1] 张锐 李德雪[1] 文兴豪[1] 张学明[1] 岳占碰[1] 于卫东[1] 

机构地区：[1]解放军军需大学动物科技系,吉林长春130062

出　　处：《中国兽医学报》2002年第4期339-340,共2页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目 (3 9770 5 5 4)

摘　　要：在含 10 %小牛血清 (NBS)的 DMEM培养基中 ,分别各添加 5 %、10 %、15 %、2 0 %和 2 5 %的二甲基亚砜(DMSO)、丙二醇 (PG)、乙二醇 (EG)和甘油 (G) ,对成年小鼠睾丸生殖细胞进行冷冻保存 ,复苏后台盼蓝染色测定细胞复苏率。结果显示 ,5 %～ 2 5 % DMSO冻存液组的细胞复苏率分别为 88.5 %、88.0 %、88.0 %、6 5 .6 %及 5 1.3% ;5 %～ 2 5 % PG冻存液组的细胞复苏率分别为 87.2 %、86 .4 %、79.0 %、73.4 %及 4 0 .1% ;5 %～ 2 5 % EG冻存液组的细胞复苏率分别为 86 .6 %、80 .9%、6 0 .8%、5 1.3%及 30 .0 % ;5 %～ 2 5 % G冻存液组的细胞复苏率分别为 86 .5 %、86 .3%、6 5 .3%、36 .0 %及 31.4 %。其中各抗冻剂 5 %和 10 %组的细胞复苏率最高 ,与 15 %组相比均存在显著或极显著差异。 4种抗冻剂的最高细胞复苏率之间无显著差异。DMSO、PG、EG和 G分别冷冻保存成年小鼠睾丸生殖细胞时的最小损失率分别为 4 .8%、6 .1%、6 .7%、6 .8%。结果表明 ,采用慢速冷冻时 ,DMSO、PG、EG及 G均适宜用作成年小鼠睾丸生殖细胞的抗冷冻剂 ,最佳使用含量均为 5 %～ 10 %。成年小鼠睾丸生殖细胞分别在含 5 %～ 10 %的 DMSO、PG、EG和 G的 DMEM(含 10 % NBS)冻存液中 ,2步慢速降温 ,液氮储存 ,37℃水浴复苏 。Adult mouse testicular germ cells were cryopreserved,and the cell recovery rates were examined by trypan blue exclusion staining after thawing.The freezing solutions were DMEM/10% NBS supplemented with 5%,10%,15%,20% and 25% cryoprotectants dimethyl sulphoxide(DMSO),propanediol(PG),ethylene glycol(EG) or glycerol(G),respectively.The cell recoveries of DMSO at concentrations of 5%,10%,15%,20%,and 25% were 88 5%,88 0%,80 0%,65 6%,and 51 3%,respectively.The cell recoveries of PG at concentrations of 5%,10%,15%,20%,and 25% were 87 2%,86 4%,79 0%,73 4%,and 40 1%,respectively.The cell recoveries of EG at concentrations of 5%,10%,15%,20%,and 25% were 86 6%,80 9%,60 8%,51 3%,and 30 0%,respectively.The cell recoveries of G at concentrations of 5%,10%,15%,20%,and 25% were 86 5%,86 3%,65 3%,36 0%,and 31 4%,respectively.When the concentrations of four cryoprotectants were 5% 10%,the cell recoveries all were highest,and showed significant difference as compared with those at concentration of 15%.There were no significant differences among the highest recoveries of four cryoprotectants.The minimum cell losses of four cryoprotectants after freezing/thawing were 4 8%,6 1%,6 7%,and 6 8%,respectively.The results suggest that DMSO,PG,EG and G all are suitable for cryopreservation of adult mouse germ cells and their favorable concentration all are at 5% 10%.The two step freezing procedure is one with high cell recovery rates for cryopreservation of adult mouse germ cells.

关 键 词：雄性生殖细胞 冷冻保存 小鼠 抗冻剂 

分 类 号：S814.3[农业科学—畜牧学]