不同培养条件对牛精原干细胞增殖的影响与特性鉴定

Effect of Different Culture Condition on Bovine Spermatogonial Stem Cells Proliferation and Characteristic Identification

探索血清浓度以及干细胞因子(SCF)、白血病抑制因子(LIF)、胶质细胞源性神经营养因子(GDNF)对牛精原干细胞体外增殖的影响,并对其进行鉴定。试验1:以高糖DMEM为基础培养液,添加浓度为0%、2.5%、5%和10%的胎牛血清(Fetal bovine serum,FBS),分为4个试验组。结果表明,血清浓度影响牛精原干细胞的体外增殖,添加2.5%的FBS比较合适。试验2:培养液中加入不同浓度的SCF、LIF及GDNF,可增加精原干细胞的数量,其中加入20μg/L SCF、80μg/L GDNF、10μg/L LIF后,与对照组比较能显著提高精原干细胞数(P<0.05)。采用碱性磷酸酶染色、细胞免疫组织化学染色和RT-PCR对牛精原干细胞进行鉴定,结果碱性磷酸酶染色呈弱阳性;细胞免疫组织化学染色Integrinβ1阳性、c-kit阳性;经RT-PCR扩增,获得了120 bp的Gfrα1序列和280 bp的c-kit序列,与预期的产物大小一致。因此,培养液中添加2.5%FBS、20μg/L SCF、80 ng/mL GDNF、10μg/L LIF对精原干细胞的增殖有利。

The aim of this paper was to study the effects of serum, SCF, LIF and GDNF on proliferation of bovine spermatogonial stem cells（SSC） in vitro, the identification of SSC was also conducted. Experiment 1： Adding 0%, 2.5%, 5% and 10% fetal bovine serum（FBS）, respectively, to the basal culture medium DMEM （Dulbecco minimal essential medium） with high glucose. The results showed that DMEM with 2.5 % FBS was mostly proper for the proliferation of bovine SSC in vitro. Experiment 2 ：Adding SCF,LIF or GDNF to the medium could increase the number of SSCs. However, only the concentrations of SCF,LIF and GDNF 20 ,80 , 10 ug/L, respectively,could significantly increase the number of SSCs compared to the control group（P〈 0.05）. Alkaline phosphatase （AP） staining , immunohistochemical staining and RT-PCR were conducted to identify bovine SSC. The results showed that AP staining was weakly positive ; Integrin β1 and c-kit staining were both positive. The sequences of Gfrα1 and c-kit were also obtained by RT-PCR, the sizes（120 bp and 280 bp） were identical to the expected sizes. Therefore,DMEM with 2.5% FBS ,adding 20μg/L SCF,80 ng/mL GDNF and 10 μg/L LIF,respectively, was appropriate for bovine SSCs culture in vitro.