大鼠精原干细胞的分离纯化

Separation and purification of rat spermatogonial stem cells

目的:探讨分离纯化大鼠精原干细胞(SSCs)的方法。方法:取大鼠睾丸组织,通过①机械法+两步酶法,②机械法+两步酶法+Percoll分离法,③机械法+两步酶法+Percoll分离法+差速贴壁法得到细胞悬液,进行培养观察。采用免疫组织化学方法鉴定,台盼蓝排斥实验及流式细胞仪分别检测细胞活率与纯度。结果:①、②、③法所得细胞为SSCs,所得SSCs活率分别为(93.26±1.06)%,(93.50±0.85)%和(94.73±0.82)%,差异无统计学意义(P>0.05);①、②、③法所得SSCs纯度呈增高趋势(P<0.05)。结论:3种方法在分离纯化过程中对SSCs活率的影响无明显差异。①法能够分离出较高纯度的SSCs,结合Percoll分离法能够使SSCs纯度提高,再结合差速贴壁法能够进一步富集SSCs。

Aim ： To discuss the separation and purification methods of rat spermatogonial stem cells （SSCs）. Methods ：The rat testicle tissue was collected. Cell suspension was isolated respectively through the following methods：method Ⅰ ： mechanical method plus two-step enzymatic method ; method Ⅱ ： mechanical method plus two-step enzymatic method plus Percoll separation method; method m ： mechanical method plus two-step enzymatic method plus Percoll separation method plus differential attachment method,respectively,and then cultured and observed. Immunohistochemistry was used to identify the SSCs. The rate of survival cells was tested by trypan blue exclusion and the purity of SSCs was evaluated by flow cytome- try. Results：The isolated cells obtained by the three methods were SSCs. There was no statistical difference in the rate of survival cells; the purity of SSCs obtained by the method Ⅱ and Ⅲ was higher than that by method Ⅰ,and the method Ⅲ was higher than the method Ⅱ, and there were statistical differences （P 〈 0.05）. Conclusion ： The rate of survival cells obtained by the 3 methods has no significant difference（ P 〉0.05 ）. The purity of SSCs obtained by method Ⅰis relatively high,when combining with Percoll separation,the purity is higher,and SSCs can be further enriched by differential attachment method.