胶质细胞源性神经营养因子对精原干细胞促进作用机制的研究

Influence mechanism of glial cell line-derived neurotrophic factor on the proliferation of spermatogonial stem cells

目的 探讨胶质细胞源性神经营养因子（glial cell line-derived neurotrophic factor,GDNF）促进精原干细胞（spermatogonial stem cells,SSCs）增殖作用的分子机制.方法 2012年1-12月合成针对GDNF基因的多个干扰性小RNA（small interfering RNA,siRNA）,以慢病毒为载体构建GDNF的慢病毒质粒,转染由日龄5～7 d SPF级健康雄性昆明小鼠睾丸组织分离出的SSCs,筛选出GDNF基因干扰效率最高的SSCs作为实验组,以GDNF基因未干扰的SSCs作为对照组.酶联免疫法分别测定两组SSCs转染后第1～4天的吸光度A值,以比较两组的增殖率.流式细胞仪检测两组SSCs的凋亡率.逆转录PCR法检测两组SSCs中GDNF、受体酪氨酸激酶（receptor tyrosine kinases,RTKs）、Fyn和局部黏着斑激酶（focal adhesion kinase,FAK）的mRNA表达.结果 实验组转染第1～4天的吸光度A值分别为0.45 ±0.02、0.68 ±0.03、1.12±0.03、2.24±0.04,对照组分别为0.46±0.03、0.73±0.02、1.32±0.05、1.15±0.06,第3、4天两组数据比较差异有统计学意义（P＜0.05）.实验组和对照组SSCs凋亡率分别为（25.43±1.91）％和（5.61±0.16）％,差异有统计学意义（P＜0.05）.实验组和对照组SSCs中GDNF、RTKs、Fyn、FAK的mRNA表达率分别为（12.32±1.22）％和（54.25±1.34）％、（16.24±1.35）％和（45.35±1.37）％、（18.32±1.34）％和（38.37±1.55）％、（20.04±1.65）％和（43.27±1.28）％,差异均有统计学意义（P＜0.05）.结论 GDNF通过上调RTKs、Fyn和FAK的表达对SSCs的增殖起促进作用.

Objective To investigate the molecular mechanisms of glial cell derived neurotrophic factor in promoting proliferation of spermatogonial stem cell.Methods RNAi expression vectors,targeted at GDNF,were constructed and transfected into SSCs from 5 to 7 days old mice.The SSCs with highest effectiveness of GDNF interfere was set as study group.And the SSCs without GDNF interfere was considered as control group.The ELISA method was used to compare the proliferative rate between study group and control group.Flow cytometry,RT-PCR were used to detect the expression of GDNF,RTKs,Fyn and FAK＇s mRNA,and the apoptosis of SSCs.Results From 1 to 4 days after transinfection,the absorbable A value in study group was 0.45 ± 0.02,0.68 ± 0.03,1.12 ± 0.03,2.24 ± 0.04,respectively.Meanwhile,the same item in control group was 0.46 ± 0.03、0.73 ± 0.02、1.32 ± 0.05、1.15 ± 0.06,respectively （P 〈 0.05）.There were significant different between experiment groups （25.43 ± 1.91） % and control group （5.61 ± 0.16）% in the apoptosis rates of SSCs （P 〈 0.05）.Significant differences were noted between experimental group and control group（P 〈 0.05）.The mRNA expression rates of GDNF was （12.32 ± 1.22） % in study group and （54.25 ± 1.34）% in control group （P 〈0.01）.The mRNA expression rates of RTKs and Fyn and FAK in study group and control group were （16.24 ± 1.35）% vs （45.35 ± 1.37）%,（18.32 ±1.34）% vs （38.37 ± 1.55）%,（20.04 ± 1.65）% vs （43.27 ± 1.28）%,respectively （P 〈0.05）.Conclusions The glial cell line derived neurotrophic factor was important in course of SSCs＇ proliferation,which may up-regulating the expression of RTKs,Fyn and FAK.