胶质细胞源性神经营养因子对体外培养小鼠精原干细胞增殖分化作用的影响

目的 探讨胶质细胞源性神经营养因子(GDNF)对体外培养小鼠精原干细胞(SSC)增殖与分化的影响.方法 雄性昆明小鼠80只.采用Percoll密度梯度分离及差速贴壁法纯化SSC,免疫荧光染色及流式细胞检测方法鉴定.将获取的SSC随机分为实验组和对照组,以支持细胞作为饲养层培养SSC,实验组每5 ml DMEM/F12完全培养液中添加0.02 μg GDNF.酶联仪检测细胞生长情况,流式细胞仪检测细胞生长周期;采用卵泡浆内显微注射(ICSI)技术将精子细胞与卵母细胞结合,观察卵裂细胞数,体外培养3 d后行染色体数量分析.结果 添加GDNF培养的SSC第6、9、12、15天吸光度(A)值分别为0.448±0.028、0.502±0.062、0.556±0.045、0.621±0.072,与对照组0.377±0.053、0.402±0.071、0.432±0.019、0.461±0.037比较差异有统计学意义(P＜0.05);第3、6、9、12、15天SSC DNA合成期(S期)含量分别为20.86、26.34、31.23、37.54、28.02,与对照组1.69、1.73、2.56,4.85,1.82比较差异均有统计学意义(P＜0.05);精子细胞与卵母细胞结合后可得到含有20对染色体的配子.结论 ICSI技术可为鉴定SSC体外分化为精子细胞提供较充分的依据;GDNF能促进SSC的体外增殖与分化.

Effect of glial cell line-derived neurotrophic factor on mouse spermatogonial stem cells in vitro

Objective To investigate the effect of glial cell line-derived neurotrophic factor(GDNF)on proliferation and differentiation of mouse spermatogonial stem cells in vitro.Methods The percoll discontinue density gradient centrifugation,followed by removing contaminated somatic cells through adhesion to plastic dishes,was used to purify the spermatogonial stem cells of Kunming mice.Mouse spermatogonial stem cells were confirmed by immunofluorescence and flow cytometry.The cells were divided into control groups and exprimental groups.The spermatogonial stem cells were cultured on sertoli cells.GDNF was added to medium of DMEM/F12.The growth of spermatogonial stem cells was determined by ELISA.The cell cycle of spermatogonial stem cells was determined by flow cytometry.Sperm was allied with ovum by intracytoplasmic sperm injection(ICSI).The chromosome figure and quantity were analysed after 3 days.Results A values of spermatogonial stem cells in experimental group were 0.362±0.031,0.448±0.028,0.502±0.062,0.556±0.045,0.621±0.072 in 3,6,9,12,15 days respectively;in control group,they were 0.365±0.045,0.377±0.053,0.402±0.071,0.432±0.019,0.461±0.037 respectively.Significant differences were noted between experimental group and controI group(P＜0.05).S period's quantities of DNA synthesizing were 20.86,26.34,31.23,37.54,28.02 in 3,6,9,12,15 days respectively,while they were 1.69,1.73.2.56,4.85,1.82 in 3,6,9,12,15 days in control group.Significant differences were noted between experimental group and control group(P＜0.05).The dual DNA could be found when sperm was integrated ovum after 3 days.Conclusions ICSI could identify spermatogial stem cells and translate into spermic cells in vitro.GDNF could improve the proliferation and differenciation of the spermatogonial stem cells in vitro.