添加N-乙酰半胱氨酸对猪卵巢颗粒细胞体外培养的影响  

Effects of N-acetylcysteine on porcine ovarian granulosa cell culture in vitro

作　　者：郭晓飞 张金龙[1] 李义海[1] 董春晓 田见晖[2] 李俊杰[3] 翁士乔 张效生[1] GUO Xiaofei;ZHANG Jinlong;LI Yihai;DONG Chunxiao;TIAN Jianhui;LI Junjie;WENG Shiqiao;ZHANG Xiaosheng(Institute of Animal Husbandry and Veterinary Medicine,Tianjin 300381,China;College of Animal Science and Technology,China Agricultural University,Beijing 100193,China;College of Animal Science and Technology,Hebei Agricultural University,Baoding 071001,China;Ningbo Sansheng Biotechnology Co.,Ltd,Ningbo 315100,China)

机构地区：[1]天津市畜牧兽医研究所,天津300381 [2]中国农业大学动物科学技术学院,北京100193 [3]河北农业大学动物科技学院,河北保定071001 [4]宁波三生药业有限公司,浙江宁波315100

出　　处：《黑龙江畜牧兽医》2020年第23期59-62,共4页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家重点研发计划项目(2017YFD0501901)。

摘　　要：为了优化猪卵巢颗粒细胞体外培养体系,探索N-乙酰半胱氨酸(N-acetyl cysteine,NAC)对猪卵巢颗粒细胞生长、增殖活力及激素分泌的影响,试验以原代培养的猪卵巢颗粒细胞为研究对象,设置4个NAC梯度浓度试验组(50,100,500,1000μmol/L)和1个空白对照组培养基进行体外培养;通过全自动细胞计数仪记录细胞生长情况,利用CCK-8试剂盒进行细胞增殖活力测定,采用放射免疫法测定类固醇激素分泌水平。结果表明:体外培养的前48 h内,4个NAC梯度浓度试验组猪卵巢颗粒细胞数量显著高于空白对照组(P<0.05),且100μmol/L浓度NAC试验组的猪卵巢颗粒细胞增殖活力显著高于空白对照组(P<0.05)。体外培养48 h后,4个NAC梯度浓度试验组猪卵巢颗粒细胞数量和增殖活力与空白对照组比较均差异不显著(P>0.05)。以培养48 h猪卵巢颗粒细胞的培养基作为测定类固醇激素的对象,同样得出100μmol/L浓度NAC试验组的孕酮和雌二醇分泌量显著高于空白对照组(P<0.05)。说明NAC添加浓度为100μmol/L时有利于猪卵巢颗粒细胞体外培养前期的贴壁生长、增殖以及细胞增殖活力的提升。In order to optimize the culture system of porcine ovarian granulosa cells in vitro, and explore the effects of N-acetyl cysteine(NAC) on the growth, proliferation activity and hormone secretion of porcine ovarian granulosa cells, primary cultured porcine ovarian granulosa cells were chosen as the research object in this experiment, and 4 NAC gradient concentration test groups(50 μmol/L, 100 μmol/L, 500 μmol/L, 1 000 μmol/L) and blank control medium were adopted to culture granulosa cells in vitro. Cell growth was recorded by an automated cell counter, cell proliferation activity was measured using the CCK-8 kit, and the level of steroid hormone secretion in the culture medium was measured by radioimmunoassay. The results showed that within the first 48 hours of in vitro culture, the number of granulosa cells in the four groups with NAC was significantly higher than that in the blank control group(P<0.05), and the proliferation activity of granulosa cells in the group with 100 μmol/L NAC was significantly higher than that in the blank control group(P<0.05). After 48 hours of culture in vitro, the number and proliferation activity of granulosa cells in each NAC group were not significantly different from those in the blank control group(P>0.05). The culture medium of porcine ovarian granulosa cells for 48 hours was used as the measurement object of steroid hormones. The secretion of progesterone and estradiol in the 100 μmol/L NAC group was significantly higher than that in the blank control group(P<0.05). The present research indicated that the dosage of 100 μmol/L NAC was beneficial to the adhesion, proliferation and proliferation activity of porcine ovarian granulosa cells at the early stage of in vitro culture.

关 键 词：N-乙酰半胱氨酸 猪 卵巢颗粒细胞 体外培养 细胞增殖活力 孕酮 雌二醇 

分 类 号：S826[农业科学—畜牧学]