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

作　　者：师如意[1,2] 张秀娟[1,3] 白银山[1] 卫恒习[1] 李莉[1] 张守全[1] 

机构地区：[1]华南农业大学动物科学学院/广东省农业动物基因组学与分子育种重点实验室,广东广州510642 [2]山西医科大学基础医学院/省部共建教育部细胞生理学重点实验室,山西太原030001 [3]广东省昆虫研究所,广东广州510260

出　　处：《华南农业大学学报》2014年第2期1-5,共5页Journal of South China Agricultural University

基　　金：973计划项目(2011CB944202;2009CB941601;2010CB945001);国家科技支撑计划(2011BAD19B03)

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＆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.

关 键 词：猪精原干细胞 超微结构 冻存 

分 类 号：Q954.43[生物学—动物学]