猪精原干细胞体外培养条件的优化

Optimizing the Culture Conditions of Porcine Spermatogonial Stem Cells (PSSCs) in vitro

摘要: 以出生后1～5 d的长白公猪为主要研究对象,采用两步酶消化法获取睾丸细胞悬液,用Percoll不连续密度梯度法对猪精原干细胞(PSSCs)进行纯化,以碱性磷酸酶(AP) 染色鉴定；利用MTT法检测2种细胞因子——胶质细胞源性神经营养因子(GDNF)和白血病抑制因子(LIF)对PSSCs发育的影响.结果表明：用Percoll不连续密度梯度法获得的PSSCs占睾丸总细胞数的(8.19±0.83)%, AP染色的阳性率为(87.47±2.90)%,表明该方法可有效获取PSSCs；MTT法检测表明,20 ng·mL^-1 GDNF+1 000 U·mL^-1 LIF的组合添加对PSSCs体外增殖的效果最好.

Abstract: The testicular cells of Landrace piglets aged from 1 to 5 days were isolated by two-step enzymatic digestion method. The Percoll discontinuous density gradients method was used to purify the porcine spermatogonial stem cells (PSSCs). The effects of glial cell-line derived neurotrophic factor (GDNF) and leukemia inhibitory factor (LIF) on PSSCs proliferation were investigated by MTT method. The results showed that the method for PSSCs isolation and purification were effective. By means of MTT assay, the results indicated that LIF and GDNF factor could promote PSSCs proliferation, and the combination of 20 ng·mL^-1 GDNF and 1 000 U·mL^-1 LIF showed the strongest effects.