奶牛卵丘细胞无血清体外培养液的优化

畜牧兽医学报 ›› 2009, Vol. 40 ›› Issue (6): 934-938.

奶牛卵丘细胞无血清体外培养液的优化

王争光，俞颂东*，许梓荣

浙江大学动物分子营养学教育部重点实验室，杭州 310029

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2009-06-26 发布日期:2009-06-26
通讯作者: 俞颂东

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

ANG Zhengguang, YU Songdong*, XU Zirong

Key Laboratory for Animal Molecular Nutrition of Ministry of Education, College of Animal Sciences, Zhejiang University, Hangzhou 310029, China

Received:1900-01-01 Revised:1900-01-01 Online:2009-06-26 Published:2009-06-26
Contact: YU Songdong

摘要/Abstract

摘要： 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15 μmol·L-1的绿茶多酚，试验4组在第3组基础上添加转铁蛋白(5 μg·mL-1)、硒(5 ng·mL-1)和胰岛素(10 μg·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。

关键词: 卵丘细胞, McCoy′s5a培养液, 体外培养

Abstract: 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 (15 μmol·L-1) were supplemented 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′s5a media are normal in morphological characteristics, growth speed and karyotype. The contents of SOD and GSH and survival rate following thawing in cumulus cells cultured with optimized McCoy′s5a media are significant higher than that in cumulus cells of control. The optimized McCoy′s5a 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.

Key words: cumulus cells, McCoy′s5a medium, culture in vitro

王争光;俞颂东;许梓荣 . 奶牛卵丘细胞无血清体外培养液的优化[J]. 畜牧兽医学报, 2009, 40(6): 934-938.

ANG Zhengguang;YU Songdong;XU Zirong. Optimization of Serumfree Media for Culturing in vitro of Cow Cumulus Cells[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2009, 40(6): 934-938.

[1]	沈朋雷, 秦希玲, 尹倩, 郭振伟, 邓凯, 杜姗姗, 尹娜, 阮子芸, 石德顺, 陆凤花. 水牛卵母细胞GV/MⅡ期对应卵丘细胞miRNA差异表达分析[J]. 畜牧兽医学报, 2017, 48(5): 836-845.
[2]	沈朋雷, 秦希玲, 尹倩, 郭振伟, 邓凯, 杜姗姗, 尹娜, 阮子芸, 石德顺, 陆凤花. 水牛卵母细胞GV/MⅡ期对应卵丘细胞miRNA差异表达分析[J]. 畜牧兽医学报, 2017, 48(5): 836-845.
[3]	李鹏飞, 孟金柱, 郝庆玲, 毕锡麟, 王锴, 朱芷葳, 吕丽华. 胰岛素和FSH对体外培养猪卵泡颗粒细胞雌激素的影响[J]. 畜牧兽医学报, 2017, 48(11): 2084-2090.
[4]	李鹏飞, 孟金柱, 郝庆玲, 毕锡麟, 王锴, 朱芷葳, 吕丽华. 胰岛素和FSH对体外培养猪卵泡颗粒细胞雌激素的影响[J]. 畜牧兽医学报, 2017, 48(11): 2084-2090.
[5]	张译夫，潘阳阳，温泽星，余四九. 表皮生长因子对牦牛卵丘细胞低氧诱导因子-1α表达的影响及与凋亡的关联性分析[J]. 畜牧兽医学报, 2016, 47(6): 1154-1161.
[6]	张译夫，潘阳阳，温泽星，余四九. 表皮生长因子对牦牛卵丘细胞低氧诱导因子-1α表达的影响及与凋亡的关联性分析[J]. 畜牧兽医学报, 2016, 47(6): 1154-1161.
[7]	曹忻，邹云龙，蔡勇，周平，李明，石国庆，康相涛. 绵羊卵母细胞体外成熟过程中VEGF对DNMT3a表达的影响[J]. 畜牧兽医学报, 2016, 47(3): 484-492.
[8]	曹忻，邹云龙，蔡勇，周平，李明，石国庆，康相涛. 绵羊卵母细胞体外成熟过程中VEGF对DNMT3a表达的影响[J]. 畜牧兽医学报, 2016, 47(3): 484-492.
[9]	陈庭锋,王霄燕,李东,施青青，张蕾,邱峰龙,刘志永,庄勋,卞桂华，宋成义,李碧春. 猪精原干细胞体外分离培养及诱导分化的研究[J]. 畜牧兽医学报, 2014, 45(7): 1104-1112.
[10]	陈庭锋,王霄燕,李东,施青青，张蕾,邱峰龙,刘志永,庄勋,卞桂华，宋成义,李碧春. 猪精原干细胞体外分离培养及诱导分化的研究[J]. 畜牧兽医学报, 2014, 45(7): 1104-1112.
[11]	夏婷，师科荣，林雪彦，侯秋玲，王中华. 肽结合与游离氨基酸比例对离体乳腺组织酪蛋白合成的影响[J]. 畜牧兽医学报, 2014, 45(3): 402-409.
[12]	夏婷，师科荣，林雪彦，侯秋玲，王中华. 肽结合与游离氨基酸比例对离体乳腺组织酪蛋白合成的影响[J]. 畜牧兽医学报, 2014, 45(3): 402-409.
[13]	李鹏飞，岳文斌，庞钰莹，于雪静，黄洋，任有蛇，吕丽华. FSH和胰岛素对绵羊卵巢卵泡颗粒细胞体外培养的影响[J]. 畜牧兽医学报, 2013, 44(9): 1386-1391.
[14]	马瑞丽，张彦明，崔红杰，郭抗抗. 新生仔猪口腔黏膜上皮细胞的体外培养方法[J]. 畜牧兽医学报, 2013, 44(9): 1445-1453.
[15]	马瑞丽，张彦明，崔红杰，郭抗抗. 新生仔猪口腔黏膜上皮细胞的体外培养方法[J]. 畜牧兽医学报, 2013, 44(9): 1445-1453.