猪精原干细胞超微结构观察与体外冻存条件的优化研究
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973计划项目(2011CB944202,2009CB941601,2010CB945001);国家科技支撑计划(2011BAD19B03)


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

    Abstract:

    【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 periods in vitro. The effects of glycerine and dimethyl sulfoxide (DMSO) on PSSCs cryopreserving were investigated.【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.

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师如意,张秀娟,白银山,卫恒习,李 莉,张守全.猪精原干细胞超微结构观察与体外冻存条件的优化研究[J].华南农业大学学报,2014,35(2):1-5

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  • 收稿日期:2013-07-03
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  • 在线发布日期: 2014-02-28