大鼠A型精原细胞的体外培养

CULTURE IN VITRO OF TYPE A SPEMATOGONIAL CELLS

目的探讨大鼠A型精原细胞的分离、鉴定及体外培养的方法与技术。方法利用非连续密度梯度离心及差速贴壁法纯化A型精原细胞;用抗c-kit和抗TERT免疫组织化学法进行细胞鉴定;用含10%NBS的DMEM进行体外培养。结果Percoll分离法结合差速贴壁法最终平均大鼠的每个睾丸可得到0.614×106个A型精原细胞,锥虫蓝染色显示细胞的存活率为92.1%。c-kit和TERT免疫组织化学阳性细胞平均分别为(90.8±1.0)%和(91.7±1.2)%。在含10%NBS的DMEM条件下,A型精原细胞于培养96 h增殖达高峰。结论Percoll分离与差速贴壁法结合是纯化A型精原细胞的有效方法;c-kit和TERT免疫组织化学结合证实本实验所获得的细胞是A型精原细胞;大鼠A型精原细胞可以在体外进行增殖。

Objective To study isolation and identification and culture of rat type A spermatogonial cells in vitro. Methods Percoll discontinue density gradient centrifugation combined with different speeds of different cells adhering to dish was used to purify the type A spermatogonial cells. The c-kit and TERT special antibodies were used to identify the type A spermatogonial cells. The purified cells were cultured in vitro. Results 0.614 × 10^6 cells per testis finally were obtained and the percentage of viable cells was 92.1% by trypan blue dye exclusion test. The percentage of type A spermatogonial cells expressing c-kit and TERT were 91.7 ± 1.2% and 90.8 ±1.0% respectively. Type A spermatogonial cells could proliferate and self-renew in the DMEM containing 10% NBS. Conclusion Percoll discontinue density gradient centrifugation combined with different speeds of different cells adhering to dish is an efficiency method for isolation of rat type A spermatogonial cells. The purified cells are type A spermatogonial cells by identification of the immunohistochemistry of c-kit and TERT antibodies. Type A spermatogonial cells can proliferate and self-renew in vitro.