以支持细胞为饲养层培养冻存后大鼠精原干细胞研究

目的：研究冻存的精原干细胞体外分离培养的生物学特性，为今后冻存精原干细胞的批量培养和长期利用提供依据。方法：取7～8d的sD雄性大鼠，分离睾丸组织，用两步酶消化法和差速时间贴壁法，分离精原干细胞和支持细胞。以二甲基亚砜（DMSO）作冷冻保护液，将分离到的精原干细胞放置于液氮中冻存；利用体外培养体系，进行支持细胞的培养和饲养层的制备；然后将复苏的精原干细胞接种到支持细胞饲养层上培养，并利用碱性磷酸酶鉴定精原干细胞。结果：用两步酶消化法和差速贴壁法能分离到纯度较高的精原干细胞和支持细胞，冻存的SSCs解冻后能在支持细胞饲养层上贴壁、生长、增殖，碱性磷酸酶活性鉴定呈阳性。结论：冻存后精原干细胞在体外支持细胞饲养层上呈集落样生长，并能长时间维持细胞集落形态及数目，可为体外研究药物或毒物干扰精原干细胞提供细胞模型。

Culture of Cryopreserved Spermatogonial Stem Cells with Sertoli Cells as Feeder Layer

Objective:To study the biological properties of cryopreserved spermatogonial stem cells (SSCs) isolated and cultured in vitro. Methods:SSCs and Sertoli cells were isolated from testes of SD rats aged 7 to 8 days by a two-step enzyme digestion method and the differential adhesion method. SSCs in DMSO were stored in liquid nitrogen,and Sertoli cells were cultured in vitro. After resuscitated,SSCs were cultured on the feeder layer of Sertoli cells and identified by alkaline phosphatase (AKP). Rusults:High purity of SSCs and Sertoli cells could be obtained by a two-step enzyme digestion method and the differential adhesion method. After cryopreservation and resuscitation,the biological properties of SSCs were not changed,including the capacity of adhesion,growth and proliferation. SSCs were found positive for alkaline phosphatase activity. Conclusion:Cultured on the feeder layer of Sertoli cells,SSCs grew as colony with their morphology and quantity remaining for a long time. The results provided a theoretical foundation for pharmaceutical research and toxicologic study on SSCs in vitro.