猪精原干细胞超微结构观察与体外冻存条件的优化研究

Ultrastructure of porcine spermatogonial stem cells( PSSCs) cultured in vitro and optimization of cryopreserving conditions

【目的】检测猪精原干细胞(PSSCs)在分化过程中其内部超微结构及细胞器的变化情况,并且找到适宜PSSCs的冷冻保存条件,为长期保存PSSCs和研究PSSCs的分化机制提供科学依据.【方法】采用两步酶消化法和Percoll不连续密度梯度法获取PSSCs,利用透射电子显微镜(TEM)对PSSCs在体外不同培养时间的超微结构变化进行观测,并且利用台盼蓝染色法和碱性磷酸酶(AP)染色法比较不同浓度的甘油和二甲基亚砜(DMSO)保存液对PSSCs冻存效果的影响.【结果和结论】未分化的PSSCs细胞膜完整平滑,细胞器正常,胞质均匀,无伪足出现,细胞核清晰.而开始分化的PSSCs出现伪足,线粒体数量大幅增加.冻存7 d后,解冻结果表明,PSSCs以V(DMSO)∶V(DMEM/F12)∶V(FBS)∶V(100×双抗)=10∶69∶20∶1为宜.

[Objective]The aim of this research was to optimize the cryopreserving conditions of porcine spermatogonial stem cells （ PSSCs ） and observe the changes of ultrastructure in PSSCs .[Method]The testicular cells of Landrace piglets aged from 1 day to 5 days were isolated by two-step enzymatic digestion method.The percoll discontinuous density gradients method was used to purify PSSCs .The transmission electron microscope （ TEM） was used to observe the ultrastructure of PSSCs during different culturing pe-riods in vitro.The effects of glycerine and dimethyl sulfoxide （ DMSO） on PSSCs cryopreserving were in-vestigated .[Result and conclusion]The results showed that the cell membrane of undifferentiated PSSCs was integrated and smoothed without pseudopodia , the cytoplasm was homogeneous and nucleus was clear.The pseudopodia were formed in differentiated PSSCs and the number of the mitochondria greatly＆amp;nbsp;increased.The appropriate cryopreserving medium for PSSCs is V（ DMSO）∶V（ DMEM/F12）∶V（ FBS）∶V（100 ＆#215;Penicillin/Streptomycin solution ）=10∶69∶20∶1.