改良培养基对体外培养生精细胞增殖分化的影响  

Proliferation and differentiation effects of modified culture medium on in vitro cultured spermatogenic cells in mice

作　　者：宋小敏[1] 姜宏[1] 汪存利[1] 黄静[1] 

机构地区：[1]解放军105医院生殖中心,合肥230031

出　　处：《安徽农业大学学报》2014年第1期14-19,共6页Journal of Anhui Agricultural University

基　　金：南京军区医学科技创新项目(09MA040)资助

摘　　要：探讨改良培养基对体外培养小鼠生精细胞的增殖分化效应。根据培养基的不同分为：A组（基础培养液组）；B组[基础培养液＋胶质细胞神经营养因子（GDNF）＋表皮生长因子（EGF）组]；C1、C2和C3组[A组＋20％、40％、80％睾丸液（TA）1；D1、D2和D3组（B组＋20％、40％、80％TA）。采用上述8种培养基对7～8日龄小鼠生精细胞进行体外培养，通过细胞形态学观察、细胞存活率和粗线期精母细胞特异性基因P19、单倍体精子细胞特异性基因TP，检测及染色体倍性分析，探讨TA、GDNF和EGF对小鼠体外培养生精细胞的增殖分化效应，结果如下：（1）形态学观察表明，B组生精细胞生长速度快，形态好，细胞集落多，A组最差；C组和D组细胞传代后培养时间最长，可达4个月。（2）生精细胞存活率结果显示，培养20d的B组细胞存活率显著高于其他各组（P〈0．05），C、D组在各培养阶段与A组间无显著性差异。（3）TP1／P19培养15d后B组的TP1／P19显著高于其他各组（P〈0．05），其他各组间均无显著性差异。（4）单倍体精子细胞比例结果表明，培养15d后的B组单倍体细胞比例显著高于其他各组（P〈0．05），其他各组间单倍体细胞比例均无显著性差异。GDNF和EGF可显著提高生精细胞体外培养的增殖分化效应，而睾丸液可显著延长生精细胞体外培养时间。The aim of the study was to investigate the proliferation and differentiation potential of juvenile mouse testis spermatogenic cells in vitro under modified culture mediums. Based on the difference of culture mediums, eight groups were set： group A （basic culture medium）, group B （basic culture medium ＋ EGF ＋ GDNF）, group C1, C2 and C3 （basic culture medium combined with 20%, 40% and 80% testicular abstract （TA））, and group D1, D2 and D3（group B combined with 20%,40% and 80% TA）. Mixed spermatogenic cells of 7-8 days old mouse were cocultured with those cultures. The proliferative and differential effects of TA, GDNF and EGF on mouse spermatogenic cells in vitro were evaluated by detecting growth behaviors and viability of sperrnatogenic cells, chromosome ploidy distribution as well as the expression of pachytene-specific phosphoprotein gene （P19） and haploid sperm cell-specific transition protein gene （TP1）. The results were as follows： （1） Through morpho- logical observation, the growth velocity of spermatogenic cells and cell colonies was best in group B, while- worst in group A; culture duration of spermatogenic cell after passage was longer in group C and group D when compared with group A and group B. （2） Survival rate of spermatogenic cell was significantly higher in group B than that in other groups after culture for 20 days, which showed no significant difference among group A, group C and group D at all culture stages. （3） The TP1/P19 in group B was significantly higher than in other groups after culture for 15 days （P〈0.05）, which showed no significant difference among other groups. （4）The proportion of haploid sperm cells in group B was significantly higher than in other groups after culture for 15 days （P〈0.05）,which showed no significant difference among other groups. In conclusion, GDNF and EGF could significantly improve proliferation and differentiation of spermatogenic cells, and TA could significantly increase the culture duration

关 键 词：生精细胞 体外培养 GDNF EGF 

分 类 号：S865.13[农业科学—野生动物驯养] Q953[农业科学—畜牧兽医]