山羊毛囊外根鞘细胞系的建立  

Establishment of Outer Root Sheath Cell Line of Goats

作　　者：崔志峰[1] 于会国[1] 张忠[1] 王慧[2] 曾勇庆[2] 

机构地区：[1]山东科技职业学院,潍坊261053 [2]山东农业大学动物科技学院,泰安271018

出　　处：《动物营养学报》2016年第10期3207-3216,共10页CHINESE JOURNAL OF ANIMAL NUTRITION

基　　金：山东省优秀中青年科学家科研奖励基金"山羊毛囊发育和毛发再生机制与毛囊体外重塑研究"(2014BSB09001);山东省农业产业创新体系虾蟹类创新团队项目(SDAIT-15-011-02)

摘　　要：本试验旨在建立纯一、稳定的山羊毛囊外根鞘细胞系。活体采集济宁青山羊羔羊背部皮肤,采用机械分离与酶消化相结合的方法,分离外根鞘细胞,培养于含有表皮生长因子（EGF）、类胰岛素生长因子-Ⅰ（IGF-Ⅰ）和氢化可的松的无血清角质细胞培养液中,置5%CO2浓度的37℃培养箱中启动原代培养。待原代细胞长成良好的单层后即可进行传代培养,细胞经传代培养至第8-10代时,更换含EGF、IGF-Ⅰ、氢化可的松和FBS的DMEM/F12培养液进行长期培养。选取传至第40代的外根鞘细胞进行生长特性研究与染色体分析。结果表明：体外培养的细胞倍增时间为51.9 h,培养细胞的染色体数仍以2n=60为主,但是出现了非整倍体性染色体特征;免疫细胞化学鉴定结果表明,该细胞系角蛋白19表达呈阳性。结果提示,本试验分离培养的细胞确为由山羊毛囊干细胞分化来的外根鞘细胞,体外培养的山羊毛囊外根鞘细胞系得到了成功建立。This study was conducted to establish a pure and genetically stable outer root sheath cell（ORSC） line of goats.Dorsal skin of lambs of Jining grey goats was collected under living condition.The method of mechanical separation conjugation with enzyme digestive was conducted to obtain ORSCs.ORSCs were cultured in keratinocyte-serum free medium with epithelial growth factor（EGF）,insulin-like growth factor-Ⅰ（IGF-Ⅰ）,and hydrocortisone for primary culture under the conditions of 5 % carbon dioxide and 37 ℃.After primary cells spread the culture flask,the subculture was started.The cells at passages 8 to 10 were then cultured in DMEM/F12 with EGF,IGF-Ⅰ,hydrocortisone and FBS for long-term culture.Growth characteristic and karyotype analysis were carried out using passage 40 cells.The results showed that the in vitro cultured cell population cost 51.9 h to perform doubling,the most of chromosome type was 2 n = 60 type,but appeared the uncompleted chromosome; the immunocytochemecal results showed that cytokeratin-19 was positively expressed in the cell line.In conclusion,the cells separated and cultured in the present study were ORSCs that differentiated from stem cells of hair follicle of goats,and a ORSC line of goats in vitro culture was successfully established.

关 键 词：毛囊外根鞘细胞 细胞系 济宁青山羊 生长特性 染色体数目 

分 类 号：S826[农业科学—畜牧学]