小鼠精原细胞体外存活、增殖特性及冷冻保存研究

为研究小鼠精原细胞体外存活、增殖特性及其以单细胞或处于功能性结构中(曲细精管或完整睾丸)耐受冷冻-解冻程序的能力。本研究收集生后7d昆白系小鼠睾丸，一步酶法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、KSOM（均含10%的FBS）后系统观察了精原细胞的存活和增殖情况；对生精上皮单细胞进行了-70℃冷冻保存和液氮冷冻保存，对曲细精管及完整睾丸进行了-70℃冷冻保存。结果显示，D-MEM和D-MEM/F12均能使小鼠精原细胞存活和增殖，精原细胞分别在培养的第1~9d或第1~8d 呈逐渐减少趋势，第9~13d或第8~12d呈增殖趋势，而第17d或第16d以后增殖减缓，KSOM只能使小鼠精原细胞短暂存活而不能使其增殖。精原细胞单独培养时增殖不明显而逐渐呈现出分化迹象。生精上皮单细胞、曲细精管及完整睾丸-70℃短期(1wk和2wk)冷冻保存或单细胞悬液液氮短期(2wk)和中长期(1个月和3个月)冷冻保存后均可以获得较理想的细胞存活率(均大于85%)。

The research of spermatogonia in vitro surviving and proliferating characteristics and cryopreservation in mouse

To investigate in vitro surviving and proliferating characteristics of mouse spermatogonia and its ability to endure freezing-thawing procedure by single cells or in their functional structure (seminiferous tubes or testes). Kun ming mouse testes of 7 day of age were collected to isolate the seminiferous epthelial cells (spermatogonia and Sertoli cells mainly) using one step of enzymatic digestion. After being plated D-MEM, D-MEM/F12 or KSOM (each contained 10% FBS) separately, the spermatogonial survival and proliferation in vitro was tracked systematically. The seminiferous epthelial cells were stored at -70℃ and liquid nitrogen, the seminiferous tubes and testes were stored at -70℃. The results indicated mouse spermatogonia could survive and proliferate in D-MEM, D-MEM/F12. Spermatogonia decreased gradually from 1d to 9d or 1d to 8d, increased rapidly from 9d to 13d or 8d to 12d in primary culture, while the increase slowed down after 17d or 16d. Mouse spermatogania could survive a short period but couldn’t proliferate in KSOM. No visible proliferation but differentiating characteristics was observed when mouse spermatogia cultured alone. The ideal percentage of viable cells (all above 85 percent) was got after the seminiferous epthelial cells, seminiferous tubes and testes stored at -70℃ for 1 or 2 weeks or the seminiferous epthelial cells stored in liquid nitrogen for 2 weeks, 1month and 3 months.