小鼠精原细胞体外增殖与冷冻保存  

In vitro Proliferation and Cryopreservation of Spermatogonia in Mouse

作　　者：李学家[1] 王勇胜[1] 刘凤军[1] 刘军[1] 张涌[1] 

机构地区：[1]西北农林科技大学生物工程研究所,杨凌712100

出　　处：《农业生物技术学报》2007年第4期633-639,共7页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.39830280);国家高技术研究发展计划(863)项目(No.2001AA213081)资助

摘　　要：利用一步酶法将7日龄昆白系小鼠睾丸分离获得生精上皮单细胞悬液(主要含精原细胞和支持细胞),分别接种于D-MEM、D-MEM/F12和KSOM后,系统研究其精原细胞的存活和增殖情况,并对生精上皮单细胞、曲细精管及完整睾丸进行了-70℃冷冻保存,同时对生精上皮单细胞进行了液氮冷冻(-196℃)保存。结果表明,小鼠精原细胞在D-MEM和D-MEM/F12中均能存活并增殖,表现为在培养的第1～9天/1～8天呈逐渐减少趋势,第9～13天/8～12天呈增殖趋势,第17天/16天以后增殖减缓,而在KSOM中只短暂存活不能增殖。生精上皮单细胞、曲细精管及完整睾丸-70℃短期(1周和2周)冷冻保存及单细胞悬液液氮短期(2周)和中长期(1和3个月)冷冻保存后,均可获得较理想的细胞存活率(均大于85%)。Kunming mouse testes of 7 days of age were collected to isolate seminiferous epthelial cells （spermatogonia and Sertoli cells mainly） using one step of enzymatic digestion, after being inoculated into D-MEM, D-MEM/F12 or KSOM ,respectively. Spermatogonial survival and proliferation in vitro were studyed systematically, and seminiferous epthelial cells, seminiferous tubes and whole testes were stored at -70℃, meanwhile, seminiferous epthelial cells were stored in liquid nitrogen. The results indicated mouse spermatogonia could survive and proliferate in D-MEM and D-MEM/F12, and they decreased gradually from 1 to 9 d or 1 to 8 d, increased rapidly from 9 to 13 d or 8 to 12 d in primary culture, the increase slowed down after 17 d or 16 d, however, mouse spermatogania could survive a short period but could not proliferate in KSOM. The ideal percentage of viable cells （all above 85 percent） was got after seminiferous epthelial cells, seminiferous tubes and testes stored at -70℃ for 1 or 2 weeks or seminiferous epthelial cells stored in liquid nitrogen for 2 weeks, 1 month and 3 months.

关 键 词：小鼠 精原细胞 增殖 冷冻保存 

分 类 号：S185[农业科学—农业基础科学]