猪精原干细胞超微结构观察与体外冻存条件的优化研究

摘要

[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 increased.The appropriate cryopreserving medium for PSSCs is V( DMSO)∶V( DMEM/F12)∶V( FBS)∶V(100 ×Penicillin/Streptomycin solution )=10∶69∶20∶1.