树鼩原代皮肤成纤维细胞的分离培养技术
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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 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.
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.
引文
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周结学,刘东,郑克立,等.2008.鼠胚成纤维细胞的培养条件及滋养层制备.中国组织工程研究与临床康复,12(34):6607-6611.
Choi E W,Seo M K,Woo E Y,et al.2017.Exosomes from human adipose derived stem cells promote proliferation and migration of skin fibroblasts.Experimental Dermatology,2017:1-3.[J/OL][2017-9-23].http://www.wileyonlinelibrary.com/journal/exd.
Fan Y,Huang Z Y,Cao C C,et al.2013.Genome of the Chinese tree shrew.Nature Communications,4:1426.doi:10.1038/ncomms2416.
Gentile D,Lazzerini P E,Gamberucci A,et al.2017.Searching novel therapeutic targets for Scleroderma:P2X7-Receptor is up-regulated and promotes a fibrogenic phenotype in Systemic sclerosis fibroblasts.Frontiers in Pharmacology,8:638.doi:10.3389/fphar.2017.00638.
Kocic H,Arsic I,Stankovic M,et al.2017.Proliferative,anti-apoptotic and immune-enhancing effects of L-arginine in culture of skin fibroblasts.Journal of Biological Regulators&Homeostatic Agents,31(3):667-672.
Kuo Y H,Wu P Y,Chen C W,et al.2017.N-(4-bromophenethyl)caffeamide protects skin from UVB-induced inflammation through MAPK/IL-6/NF-kappaB-Dependent signaling in human skin fibroblasts and hairless mouse skin.Molecules,22(10):1-14.
Tsukiyama-Kohara K,Kohara M.2014.Tupaia belangeri as an experimental animal model for viral infection.Experimental Animals,63(4):367-374.
Wu Z,Zhang L.2017.Polycomb group proteins:Novel molecules associated with ultraviolet A-induced photoaging of human skin.Experimental and Therapeutic Medicine,14(3):2554-2562.
Zhou X,Sun F,Xu S,et al.2015.The position of tree shrews in the mammalian tree:Comparing multi-gene analyses with phylogenomic results leaves monophyly of Euarchonta doubtful.Integrative Zoology,10(2):196-198.
陈瑾,代解杰,孙晓梅.2008.树鼩肝炎动物模型的研究进展.中国比较医学杂志,18(2):59-62.
崔照琼,杨卫,李艳琼,等.2016.树鼩HSV-1感染模型的建立.中国病原生物学杂志,11(4):325-333.
洪城,王健,李冰,等.2010.分次胶原酶消化法分离、培养成人肺微小动脉平滑肌细胞.中国病理生理杂志,26(6):1240-1243.
胡素贤,倪晓妍,王嘉逊,等.2011.新生小鼠皮肤成纤维细胞的分离培养与鉴定.中国医药导报,8(29):5-7+26+193.
李雅钗,黄向华,宋淑霞.2010.组织块法和酶消化法体外培养原代鼠阴道上皮细胞的研究.中国细胞生物学学报,32(5):758-761.
徐林,张云,梁斌,等.2013.实验动物树鼩和人类疾病的树鼩模型研究概述.动物学研究,34(2):59-69.
赵志敏,王国坤,王杨,等.2017.胶原酶消化法、组织块贴壁法培养的大鼠RASMCs生长、增殖及分化对比观察.山东医药,57(28):33-36.
周结学,刘东,郑克立,等.2008.鼠胚成纤维细胞的培养条件及滋养层制备.中国组织工程研究与临床康复,12(34):6607-6611.