小鼠精原干细胞冷冻保存  

Cryopreservation of mouse spermatogonial stem cells

作　　者：丁晓麟[1] 张寒莹[1] 王子玉[1] 张艳丽[1] 许欣[1] 石国庆[2] 王锋[1] 

机构地区：[1]南京农业大学动物胚胎工程技术中心,南京210095 [2]新疆农垦科学院畜牧兽医研究所,石河子832000

出　　处：《解剖学报》2009年第4期685-689,共5页Acta Anatomica Sinica

基　　金：教育部“春晖计划”资助项目(z2004-2-65019)

摘　　要：目的探索小鼠精原干细胞（SSCs）冷冻保存方法以及解冻后体外快速增殖的条件。方法实验以6d龄雄性昆明小白鼠为材料,两步酶消化法分离小鼠睾丸生殖细胞,Percoll非连续密度梯度离心法富集小鼠精原干细胞,随后加入不同的冷冻液以及采用不同的降温速率冷冻小鼠精原干细胞。以MEMα为基本培养基,加入10%胎牛血清和100μg/L的胶质细胞源性神经营养因子,WST-8比色法分析培养SSCs复苏后的增殖率,运用碱性磷酸酶细胞化学染色和RT-PCR技术,对培养的SSCs进行鉴定。结果冷冻液中添加10%二甲基亚砜、10%胎牛血清0、.07mol/L蔗糖时,以1℃/min程控降温方式冷冻小鼠SSCs,细胞解冻后活率最高,达84%以上;采用非程控降温方式冻存SSCs,尽管细胞解冻后活率相对于程控降温方式略低,但具有方法简单、易于操作、无需昂贵仪器的优点,复苏后SSCs贴壁时间为8~12h,24h可见细胞分裂,48h细胞出现迅速增殖,96h可见较多含20~25细胞的细胞团,此时精原干细胞增殖近5倍。结论本实验所用培养条件,可以使经长期冷冻的SSCs短期快速增殖。Objective To explore the conditions and methods for cryopreservation and proliferation of mouse spermatogonial stem cells （SSCs）. Methods SSCs were isolated from six-day-old Kunming mouse using two-step enzymatic digestion and Percoll discontinuous density gradient centrifugation. Cells were frozen with different freezing medium and cooling rate. After thaw, they were cultured in mimimum essential medium alpha （MEMu） supplemented with 10% fetal calf serum （FCS） and 100ttg/L glial cell line-derived neurotrophic factor （GDNF）. The survived and proliferating SSCs were examined by WST-8 colorimetfic assay. Alkaline phosphatase and reverse transcription-polymerase chain reaction （RT-PCR） were performed to confirm if the cultured 96 hours germ cells were still stem ceils. Results The best method to eryopreserve SSCs is using eryoprotector containing 10 % dimethyl sulfoxide （ DMSO）, 10% FCS, 0.07mol/L sucrose and 1 ~C/rain cooling rate, and the viability of cells in this method is more than 84 % ; Although the cell viability in non-programmed freezing method is less than that in the programmed freezing method, it is a simple and effective cropreservafion method for mouse SSCs. What is more, the anchoring time of SSCs in this method is 8-12 hours after thaw, SSCs begin to proliferate 24 hours later, and rapid proliferation appears on the 48 hours, colonies are composed by 20-25 cells in 96 hours, when SSCs proliferated nearly 5 times. Conclusion The culture condition we used is suitable for proliferation of frozen-thawed SSCs.

关 键 词：冷冻保护剂 降温速率 细胞培养 精原干细胞 小鼠 

分 类 号：Q785[生物学—分子生物学]