树鼩原代皮肤成纤维细胞的分离培养技术  

Isolation and Culture of Primary Skin Fibroblasts from Tree Shrews(Tupaia belangeri)

作　　者：白剑 焦玲帅 杨铭[1] 王武 代解杰[2] 夏雪山[1] BAI Jian;JIAO Ling-Shua;YANG Ming;WANG Wu;DAI Jie-Jie;XIA Xue-Shan(Faculty ofLife Science and Technology,Kunming University of Science and Technology,Kunming 650500;Yunnan ProvincialInnovation Group of Tree Shrew Standard Culture and Animal Model,Institute of Medical Biology,Chinese Academy of Medical Sciences andPeking Union Medical College,Kunming 650118,China)

机构地区：[1]昆明理工大学生命科学与技术学院,昆明650500 [2]中国医学科学院/北京协和医学院医学生物学研究所树鼩标准化研究与疾病动物模型省创新团队,昆明650118

出　　处：《动物学杂志》2019年第1期76-86,共11页Chinese Journal of Zoology

基　　金：国家科技支撑计划项目(No.2014BAI01B01)

摘　　要：建立稳定的树鼩(Tupaiabelangeri)皮肤成纤维细胞的体外培养体系,可为有关此类细胞的实验和疾病树鼩细胞模型提供技术支持。取树鼩大腿内侧皮肤用组织块贴壁法和胶原酶Ⅰ消化法分离皮肤细胞,胰蛋白酶差别消化法纯化细胞;用MEM(10%FBS)完全培养基和含低血清生长添加物(LSGS)的培养基培养细胞;免疫荧光和蛋白印迹法鉴定细胞,并测定细胞的生长、冻存和复苏特性。经树鼩皮肤细胞分离效果比较,胶原酶消化法比组织块贴壁法更适合用于树鼩原代皮肤细胞分离;对分离及冻存复苏后细胞生长状况观察比较发现,添加了LSGS的MEM培养基更利于细胞存活、生长;细胞形态观察、免疫荧光和蛋白印迹检测鉴定所分离的细胞为树鼩皮肤成纤维细胞。成功建立了树鼩原代皮肤细胞的分离、纯化方法,并优化了该细胞的培养条件。To establish a stable culture system of skin fibroblasts from Tree Shrew(Tupaia belangeri),and provide a primary cell model for related experiment on this specific animal species,the inner thigh skin was scraped to isolate primary cells using tissue mass adherence and collagenaseⅠdigestion,respectively.The obtained cells were purified by using differential digestion with trypsin.MEM(with 10%FBS)and low Serum Growth Supplement added MEM(LSGS)were used for cell culture.The cells were identified by immunofluorescence and Western blot analysis,and the characteristics of cells′survival and replication were further assessed.The results showed that collagenase digestion was more suitable for isolation of primary skin cells than method of tissue adherence(Fig.1,2).According to the survival and growth characteristics of isolated cells(Fig.5,6),the LSGS may provide a better culture condition for isolated cells in comparing with that of MEM(Fig.4).Immunofluorescence observation(Fig.8 and Fig.9)and Western blot(Fig.10)detection revealed that the isolated cells were primary skin fibroblasts.This effective method for isolation of primary skin fibroblasts from Tree Shrews was successfully established and their culture conditions were optimized for further investigation.

关 键 词：树鼩 原代皮肤成纤维细胞 分离 培养 鉴定 

分 类 号：Q952[生物学—动物学]