【摘要】 【目的】探讨不同血清和细胞因子等培养液添加物对尼罗罗非鱼(Oreochromisniloticus)精原干细胞(Spermatogonial stem cells,SSCs)生长增殖的影响。【方法】无菌取发育第Ⅲ期的尼罗罗非鱼精巢组织,用酶消化法结合差速贴壁法获得尼罗罗非鱼SSCs,在有支持细胞饲养层条件下,L-15培养液中分别添加体积分数2%、5%、10%新生牛血清(New bovine serum,NBS),体积分数1%、2%、3%罗非鱼血清以及5、10、15 ng/mL白血病抑制因子(Leukemia inhibitor factor,LIF),比较不同添加物对尼罗罗非鱼SSCs生长增殖的影响。【结果】在支持细胞饲养层上,SSCs分裂指数明显高于无饲养层的SSCs,支持细胞饲养层明显促进精原干细胞分裂增殖(P <0.01);与2%、10%NBS组相比,添加体积分数5%NBS可显著促进精原干细胞增殖(P <0.01);添加体积分数1%、2%、3%尼罗罗非鱼血清对SSCs无显著的促增殖作用(P> 0.05);添加5、10 ng/mL LIF可促进SSCs增殖,添加10 ng/mL LIF效果更佳,添加15 ng/mL LIF则抑制SSCs增殖。【结论】使用尼罗罗非鱼精巢支持细胞作饲养层,用添加体积分数5%NBS及10 ng/mL LIF的L-15培养液培养,有助于促进尼罗罗非鱼精原干细胞生长增殖。更多还原

【Abstract】 【Objective】To investigate the effects of different medium additives on growth and proliferation of the spermatogonial stem cells(SSCs) of Nile tilapia(Oreochromis niloticus).【Methods】The SSCs were obtained from fresh testis of Nile tilapia in the development stageⅢ by pancreatic enzyme digestion and followed differential adhesion.With the Sertoli cells as feeder layer,the growth and proliferation of SSCs were investigated by culturing in L-15 culture medium supplemented with 2%,5%,10% NBS(new bovine serum),or 1%,2% and 3% Nile tilapia serum,or 5,10 and 15 ng/mL LIF(leukemia inhibitor factor),in different combinations.【Results】The splitting index of SSCs with Sertoli cell feeder was significantly higher than those without the feeder(P<0.01).Sertoli cell feeder layer significantly could speed up division and proliforation of spermatogonial stem cells significantly.Compared to L-15 with 2% and 10% new bovine serum,proliferation of spermatogonial stem cells was respectively increased(P<0.01) in 5% new bovine serum medium.However,supplementation of 1%,2% and 3% Nile tilapia serum in culture medium had no effect on proliferation of SSCs.Proliferation of spermatogonial stem cells was increased in culture medium supplemented with 5 ng/mL and 10 ng/mL leukemia inhibitor factor and 10 ng/mL had better result,but decreased with 15 ng/mL LIF.【Conclusions】The proliferation of Nile tilapia SSCs was improved by using Sertoli cells as feeder layer and cultured in L-15 cell culture medium with 5% new bovine serum and 10 ng/mL leukemia inhibitor factor.更多还原