鸡精原干细胞分离培养初步研究

Preliminary Study on the Isolation and in vitro Culture of Chicken Spermatogonial Stem Cells

[目的]建立禽类精原干细胞(SSCs)体外培养系统。[方法]以25日龄广西土鸡为试材,通过睾丸石蜡切片和HE染色,了解SSCs发育情况,用两步酶消化法分离获得生精上皮细胞,比较不同温度、接种密度对鸡SSCs体外培养的影响,并对SSCs的集落进行染色鉴定。[结果]结果显表明,25日龄时SSCs已经形成,但还未分化。37℃适宜作为鸡SSCs的培养温度,5×105个/ml适宜作为鸡SSCs原代混合培养的接种密度。经碱性磷酸酶和糖原染色鉴定,体外培养的鸡SSCs仍保持未分化的特性。[结论]该研究为禽类SSCs体外培养的基础资料提供参考。

[Objective] The aim of study was to found in vitro culture system of SSCs for birds kind.[Method] 25-day-old male chickens were used to isolate seminiferous epithelial cells by sequential two-step enzyme digestion.The spermatogonial stem cells（SSCs） development stage was evaluated by histological examination after hematoxylin-eosin staining.The effects of different culture temperatures and concentration on primary culture of chicken SSCs were evaluated,and the SSCs colonies were characterized by staining.[Result]The results showed that by 25-days,SSCs,as the most advanced germ cells,were present,but undifferentiated.The temperatures at 37 ℃ and seeding at a concentration of 5×105 cells/ml were suitable for chicken SSCs culture.During in vitro culture,the colony-forming SSCs were stained positively for alkaline phosphatase and periodic acid schiff solution,demonstrating that these cells still remained undifferentiated in this culture system.[Conclusion]This study will provide the reference for basic data of in vitro culture of bird kind SSCs.