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白剑,焦玲帅,杨铭,王武,代解杰,夏雪山.2019.树鼩原代皮肤成纤维细胞的分离培养技术.动物学杂志,54(1):76-86.
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)
投稿时间:2017-10-12  修订日期:2018-10-22
DOI:10.13859/j.cjz.201901010
中文关键词:  树鼩  原代皮肤成纤维细胞  分离  培养  鉴定
英文关键词:Tree Shrew, Tupaia belangeri  Primary skin fibroblasts  Isolation  Culture  Identification
基金项目:国家科技支撑计划项目(No. 2014BAI01B01)
作者单位E-mail
白剑 昆明理工大学生命科学与技术学院 yrbaijian@163.com 
焦玲帅 昆明理工大学生命科学与技术学院 jiaolingshuai@163.com 
杨铭 昆明理工大学生命科学与技术学院 qjyangming@163.com 
王武 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 wangwu@163.com 
代解杰 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 djj@imbcams.com.cn 
夏雪山 昆明理工大学生命科学与技术学院 oliverxia2000@aliyun.com 
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中文摘要:
      建立稳定的树鼩(Tupaia belangeri)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。
英文摘要:
      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 collagenase Ⅰ digestion, 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.
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