赤狐皮肤黑色素细胞的分离与体外培养  

Isolation and culture in vitro of melanocytes from the skin of red fox(Vulpes vulpes)

作　　者：鲍加荣[1] 刘学庆[1] 岳志刚[1] 杨福合[1] 

机构地区：[1]中国农业科学院特产研究所/吉林省特种动物分子生物学省部共建国家重点实验室,长春130112

出　　处：《南方农业学报》2015年第8期1511-1515,共5页Journal of Southern Agriculture

基　　金：国家"973"计划项目(2012CB72907)

摘　　要：【目的】建立一种适合赤狐皮肤黑色素细胞的分离和体外培养体系,为研究毛色形成的分子机理提供必要载体。【方法】采集赤狐的皮肤样品,经DispaseⅡ和胰酶两步消化,以添加有表皮生长因子(Epidermal growth factor,EGF)、十四烷酰佛波醇乙酸酯(12-O-tetradecanoyl-phorbol 13-acetate,TPA)和牛垂体提取物(Bovine pituitary extract,BPE)的K-SFM培养基进行原代和传代培养,再通过形态观察、用酶标仪测定黑色素含量、多巴染色结合透射电镜观察、毛色基因RT-PCR检测等方法对体外培养获得的黑色素细胞进行鉴定。【结果】体外培养获得的赤狐皮肤黑色素细胞有2或3个树突,胞质内含有大量的黑色素小体,经测定第3、4和5代细胞的黑色素含量分别为0.0283、0.0276和0.0265 ng/cell,黑色素含量差异不显著(P>0.05)。从第4代赤狐皮肤黑色素细胞的总RNA中能RT-PCR扩增出MITF、TYR、TYRP1、MITF等毛色基因特异条带,说明培养获得的赤狐皮肤黑色素细胞生物学状态良好。【结论】以K-SFM为基础培养基,通过添加EGF、TPA和BPE等成分,可体外培养获得赤狐皮肤黑色素细胞,且能有效回避添加CT培养细胞的潜在致癌风险。【Objective】The present experiment was conducted to establish an efficient system for isolation and culture in vitro of melanocytes from skin of red fox（Vulpes vulpes）, in order to provide essential vector for studying molecular mechanism of coat color formation. 【Method】The skin samples were collected from adult red foxes, then the melanocytes suspension was obtained by digesting samples with Dispase II and 0.25% Trypsin-EDTA and culturing with K-SFM medium supplemented with EGF, BPE and TPA. At last, the melanocytes dyed with Dopa were identified under transmission electron microscope. And the melanin contents of the 3^rd, 4^thand 5^thgenerations melanocytes were determined by using ELIASA, respectively. Moreover, the coat color genes were detected by using RT-PCR technique. 【Result 】The results showed that the two or three dendrites and a large number of melanosomes were found in the obtained melanocytes by observing with inverted electron microscope, and the melanin contents were 0.0283, 0.0276 and 0.0265 ng/cell in 3^rd, 4^th and 5^thgenerations melanocytes, respectively, and these were no significant difference（P〉0.05）. Moreover, the MITF, TYR and TYRP1 and other specific coat color gene fragments were amplified from the 4^thgeneration melanocytes using RT-PCR technique. 【Conclusion】The melanocytes from skin of red fox can be cultured in vitro in basal medium of K-SFM supplemented with TPA, b EGF and BPE. Thereforemore, the present experiment not only lays a cytological foundation for investigating coat color formation, but also provides appropriate materials to study function of color genes and diseases related to coat color.

关 键 词：赤狐 黑色素细胞 多巴染色 透射电镜 

分 类 号：S865.23[农业科学—野生动物驯养]