麦穗鱼囊胚细胞的分离与冷冻保存  

Isolation and cryopreservation techniques of Pseudorasbora parva blastomeres

作　　者：方晨[1] 王霞[1] 王伟[1] 龚良超[1] 刘学[1] 罗昌妃 张小雪[1] 

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025

出　　处：《广东农业科学》2015年第24期124-128,F0003,共6页Guangdong Agricultural Sciences

基　　金：贵州省优秀科技教育人才省长专项资金(黔省专合字[2009]102号)

摘　　要：为了探讨麦穗鱼囊胚细胞的分离方法及其较佳的冷冻保存条件,采用胰蛋白酶酶解法和低温冷冻保存技术对麦穗鱼囊胚细胞的分离和保存进行研究。结果显示,20℃微流水条件下,受精后120 min左右麦穗鱼胚胎发育达到囊胚期;25℃条件下,0.25%胰蛋白酶作用12~18 min,麦穗鱼囊胚细胞即可脱去胚胎膜,且所获的囊胚细胞存活率可达90%以上;10%DMSO＋DMEM作为冷冻储存保护液,储存于-80℃超低温冰箱中8 d,囊胚细胞存活率保持在50%以上;储存于-196℃,8 d后囊胚细胞存活力可达70%以上,储存128 d后,细胞存活率仍可保持60%以上。表明25℃、0.25%胰蛋白酶作用是较好的麦穗鱼囊胚细胞卵膜去膜条件,10%DMSO＋DMEM保护液、细管和液氮（-196℃）保存囊胚细胞128 d可保持其有60%以上的存活率。To study the isolation and suitable cryopreservation of Pseudorasbora parva blastomeres,comparison experiments were conducted in taking off the egg membrane of P. parva blastula with different concentrations of trypsase at 25℃,and in cryopreservation for P. parva blastomeres with different protectants and at different temperatures. Results showed that the blastula stage of P. parva was achieved in 120 min after fertilization,under the condition of microstream at 20℃. It took 12-18 min to take off the egg membrane with 0.25% trypsase at 25℃,furthermore,the survival rate of isolated blastomeres was above 90%. 10% DMSO＋DMEM was used as a tprotectant at-80℃,the blastomeres survival rate was above 50% after 8 d;at-196℃,the survival rate was above 70% after 8 d,and the survival rate was also above 60% after 128 d. The result indicated that better condition for taking off the egg membrane was treatment with 0.25% trypsase for 12-18 min at 25℃. Higher blastomeres survival rate（above 60%）could be maintained at-196℃ and 10% DMSO＋DMEM.

关 键 词：麦穗鱼 囊胚细胞 分离 冷冻保存 

分 类 号：S917.4[农业科学—水产科学]