犬乳腺上皮细胞的分离鉴定及培养方法优化  

Isolation,identification and culture method optimization of Canine mammary epithelial cells

作　　者：冯子建 孙娜[1] 郝志立 孙盼盼 张华[1] 范阔海[3] 尹伟[1] 杨惠珍 钟佳 张振彪 王建中 孙耀贵[1] 李宏全[1] FENG Zijian;SUN Na;HAO Zhili;SUN Panpan;ZHANG Hua;FAN Kuohai;YIN Wei;YANG Huizhen;ZHONG Jia;ZHANG Zhenbiao;WANG Jianzhong;SUN Yaogui;LI Hongquan(School of Veterinary Medicine/Shanxi Provincial Key Laboratory of Modernization of Traditional Chinese Veterinary Medicine,Shanxi Agricultural University,Taigu 030801,China;College of Veterinary Medicine,Jilin University,Changchun 130015,China;Laboratory Animal Management Center,Shanxi Agricultural University,Taigu 030801,China)

机构地区：[1]山西农业大学动物医学学院/中兽医药现代化山西省重点实验室,山西太谷030801 [2]吉林大学动物医学学院,长春130015 [3]山西农业大学实验动物管理中心,山西太谷030801

出　　处：《中国农业大学学报》2023年第6期160-168,共9页Journal of China Agricultural University

基　　金：国家自然科学基金项目(32172938,32172904);山西省重点研发计划项目(202102140601019)。

摘　　要：为建立一种快速分离犬原代乳腺上皮细胞的培养方案,本研究旨在从健康泌乳金毛犬的乳汁中分离原代乳腺上皮细胞,接种于两种不同的培养基后传代培养,探讨不同培养基下细胞的生物学特征,提供犬乳腺上皮细胞简单便捷的分离措施及适宜的培养方法。选取泌乳一周的金毛犬在无菌条件下收集乳汁,离心处理后得到乳腺上皮细胞,分别用完全培养基和基础培养基接种培养,经形态学对比观察,间接免疫荧光检测角蛋白8(Cytokeratin 8)表达情况,绘制两种培养基培养下的第3代乳腺上皮细胞生长曲线;探讨完全培养基培养的第6代乳腺上皮细胞的冻存复苏活力。结果表明:1)乳汁分离可得到大量细胞团块,分别接种两种培养基后都会出现“煎蛋样”贴壁细胞,消化传代后两者均有Cytokeratin 8的高度表达;2)接种于基础培养基的细胞前期以聚集在一起的圆形单克隆细胞团块为主,传至第4代仅有少量细胞存活,细胞随着培养时间增加而状态变差,死亡细胞增多;接种于完全培养基的细胞整体呈“鹅卵石铺路样”成片生长,随着传代次数增加细胞状态稳定,细胞生长曲线呈现“S”形;3)完全培养基培养的第6代乳腺上皮细胞冻存复苏后能快速贴壁并分裂增殖。综上,乳汁分离法成功分离犬乳腺上皮细胞,且与基础培养基相比,完全培养基可以更好地维持细胞活性,促进细胞增殖分裂,且不影响细胞冻存后复苏的活力,为后期快速分离犬乳腺上皮细胞提供较优的培养方案。In order to establish a culture scheme for rapid isolation of primary canine mammary epithelial cells,the study was to isolate canine mammary epithelial cells from the milk of healthy lactating golden retrievers,inoculate them with two different media and subculture them,and to explore the biological characteristics of cells in different media to provide simple and convenient isolation measures and suitable culture methods for canine mammary epithelial cells.The milk of golden retrievered dogs that had been lactating for one week was collected under sterile conditions,and mammary epithelial cells are obtained after centrifugation and cultured with complete and basal medium,respectively.Through morphological comparison and observation,the expression of cytokeratin 8 was detected by indirect immunofluorescence,and the growth curve of the third generation of mammary epithelial cells cultured in two kinds of media was drawn for investigating the cryopreservation and resuscitation activity of the 6th generation mammary epithelial cells cultured in complete medium.The results showed that:1)A large number of cell clumps can be obtained by milk separation,and“fried egg-like”adherent cells appear after inoculation of both mediums,and both are highly expressed by cytokeratin 8 after digestion and passage.2)The cells inoculated in the basal medium were mainly round monoclonal cell clumps gathered together in the early stage,and only a few cells survived in the fourth generation.With the increase of culture time,the cells became worse,and grew in a“cobblestone paving”shape,and the cell state was stable with the increase of passage times,and the cell growth curve presented a“S”shape.3)After cryopreservation and recovery,the 6th genration mammary epithelial cultured in complete medium could adhere to the wall and and proliferate rapidly.In summary,the milk separation method successfully isolated the canine mammary epithelial cells.And compared with the basal medium,complete medium could better maintain cell activit

关 键 词：犬乳腺上皮细胞 乳汁分离 Cytokeratin 8 培养优化 

分 类 号：S85[农业科学—兽医学]