GDNF和LIF对小鼠精原干细胞体外增殖的影响

Proliferation effects of GDNF and LIF on mouse spermatogonial stem cells in vitro

【目的】探讨胶质细胞源性神经营养因子(GDNF)和白血病抑制因子(LIF)对小鼠精原干细胞(SSCs)体外增殖的影响,为后续SSCs的诱导分化、转基因动物生产、基因治疗等研究奠定基础。【方法】收集6～8日龄小鼠睾丸,采用机械法和2步酶消化法获得细胞悬液。通过多次差异贴壁法分离纯化SSCs和支持细胞,采用碱性磷酸酶(AP)染色和RT-PCR检测Ngn3和Oct4基因2种方法对SSCs进行鉴定。采用单独添加GDNF(添加量为0,10,20,40ng/mL)或LIF(添加量为0,500,1 000,1 500U/mL)的无血清DMEM/F12培养基培养SSCs,于培养第3,5,7天取样,同时用添加GDNF和LIF(各因子单独添加量两两组合)的无血清DMEM/F12培养基培养SSCs,于培养第3,4,5天取样,采用甲基噻唑基四唑(MTT)法检测GDNF、LIF对SSCs体外增殖的单因子效应和配伍效应。【结果】与对照组相比,不论培养时间如何,单独添加20和40ng/mL的GDNF可以显著促进SSCs增殖(P<0.05),而单独添加不同量LIF对SSCs增殖的影响不显著(P>0.05);同时添加20ng/mL GDNF和1 000U/mL LIF可以显著促进SSCs的增殖,在该条件下当精原干细胞接种密度为(6×104)～(10×104)mL-1,共培养5d时,其OD490值为0.696。【结论】DMEM/F12培养基中单独添加20ng/mL GDNF或同时添加20ng/mL GDNF和1 000U/mL LIF可以显著促进小鼠SSCs的体外增殖。

Objective】 The aim of this study was to determine the effects of GDNF and LIF on mouse spermatogonial stem cells in vitro,which lays the foundation for subsequent experiments,such as researches of pluripotency of SSCs,production of genetically modified animals,gene therapy,and so on.【Method】 Six to eight-day-old mouse testes were harvested and cell suspension was obtained by mechanical method and two-step enzyme digestion method.The SSCs and Sertoli cells were separated by differences of cells adherence.The two kinds of cells were identified by alkaline phosphatase（AP） staining and RT-PCR.Serum-free DMEM/F12 media added with GDNF（addition：0,10,20,40 ng/mL） or LIF（addition：0,500,1 000,1 500 U/mL） alone and in combination was used to culture SSCs.On the 3rd,5th,7th and the 3rd,4th,5th day.Single-factor effect and double-factor effect of GDNF and LIF on SSCs proliferation were separately tested by methyl thiazolyl tetrazolium（MTT） assay.【Result】 Compared with the control,20 and 40 ng/mL of GDNF could largely promote cell growth,regardless of the culture time（P〈0.05）.However,there was no significant difference to promote proliferation of SSCs between LIF treatment groups and the control（P〉0.05）;Adding 20 ng/mL GDNF and 1 000 U/mL LIF in combination could significantly enhance mouse SSCs proliferation in vitro（P〈0.05）,and under this condition,the OD490 value was 0.696 on 5th day culture when the concentration of SSCs was（6×104）-（10×104） cells/mL.【Conclusion】 Serum-free DMEM/F12 media added with 20 ng/mL GDNF or 20 ng/mL GDNF and 1 000 U/mL LIF in combination could significantly promote mouse SSCs proliferation in vitro.