奶牛卵丘细胞无血清体外培养液的优化

Optimization of Serum-free Media for Culturing in vitro of Cow Cumulus Cells

采集奶牛卵巢表面卵泡中的卵丘-卵母细胞复合物,通过胰蛋白酶消化获得卵丘细胞,分别利用对照组DMEM/F12培养液和4个试验组McCoy′s5a培养液对卵丘细胞进行原代及传代培养。试验1组用McCoy′s5a基础培养液,试验2组在McCoy′s5a基础培养液中添加29 ng.mL-1的睾酮,试验3组同时添加29 ng.mL-1的睾酮和15μmol.L-1的绿茶多酚,试验4组在第3组基础上添加转铁蛋白(5μg.mL-1)、硒(5 ng.mL-1)和胰岛素(10μg.mL-1),构建成优化培养液(Optimized McCoy′s5a)。结果表明,试验4组优化培养液所培养的卵丘细胞的形态和增殖规律符合正常细胞的一般规律,染色体核型正常,其细胞内SOD、GSH含量及冷冻解冻后的存活率均显著高于对照组,优化培养液可以代替含血清的DMEM/F12培养液用于奶牛卵丘细胞的体外培养。

The cumulus cells were obtained by digesting cumulus-oocyte complexes with trypsin solution, and were cultured in DMEM/F12 media （control group） or McCoy＇s5a media （four treatment groups） during primary culturing and subculturing. The McCoy＇s5a were utilized for media in treatment 1. The media for treatment 2 were McCoy＇s5a media containing 29 ng · mL-1 testosterone. Testosterone （29 ng · mL-1） and green tea polyphenols （15μmol ·L-1 were sup- plemented together into McCoy＇s5a media in treatment 3. In treatment 4, optimized McCoy＇s5a media were utilized for culturing cumulus cells. Optimized McCoy＇s5a media were supplemented with transferrin, selenium and insulin based on media in treatment 3. The results show that the cumulus cells cultured with optimized McCoy＇sSa media are normal in morphological characteris- tics, growth speed and karyotype. The contents of SOD and GSH and survival rate following tha- wing in cumulus cells cultured with optimized McCoy＇s5a media are significant higher than that in cumulus cells of control. The optimized McCoyr sSa media with appropriate concentration of transferrin, selenium and insulin can instead of DMEM/F12 media containing serum to culture in vitro cumulus cells of cow.