摘要
为探讨泰和乌骨鸡鸡胚皮肤黑色素细胞的分离、培养及鉴定方法,取20日龄的泰和乌骨鸡鸡胚的背部皮肤,通过不同的消化酶处理皮肤分离乌骨鸡黑色素细胞,细胞培养于改良型的α-MEM完全培养液中,对黑色素细胞进行形态学观察,并通过四甲基偶氮唑蓝(MTT)法观察黑色素细胞的生长情况,用多巴染色和抗S-100免疫细胞化学染色法对培养的细胞进行鉴定。结果表明,用Dispase+胰蛋白酶消化皮肤可获得较好的分离效果,倒置显微镜下观察细胞呈树突状。培养液中添加碱性成纤维细胞生长因子(b FGF)和霍乱霉素(CT)可协同促进黑色素细胞的生长。MTT结果显示细胞生长良好,多巴和抗S-100蛋白染色呈阳性。本试验成功从20日龄泰和乌骨鸡鸡胚背部皮肤中分离得到黑色素细胞,经形态学观察、多巴和免疫细胞化学染色鉴定为黑色素细胞。
The objective of this study was to develop the methods for isolation,cultivation and iden- tification of melanocytes from embryo skin of Taihe silky fowl. Skin isolated from 20-day-old Taihe silky fowl embryos was subjected to digestion with different enzymes to isolate melano- cytes. The isolated cells were cultured in modified α-MEM comprehensive medium, and then cells were subjected to morphological observation. The growth capacity of melanocytes was evaluated by methyl thiazolyl tetrazolium （MTT） method. Dopa staining and immunostaining with anti-S- 100 protein were performed to identify the cultured melanocytes. Melanocytes could be effectively obtained by digestion with Dispase plus trypsin,and the resulted cells were morphologically den- dritic as observed under inverted phase contrast microscope. Supplementation with basic fibroblast growth factor （bFGF） and cholera toxin （CT） in the culture medium synergistically promoted melanocyte replication. MTT results showed that melanocytes isolated with Dispase-trypsin digestion method grew well,and the ceils were positive for Dopa and S-100 staining. In summary, mela- nocytes were successfully obtained from backside skin of 20-day-old Taihe silky fowl embryos by above method,and the obtained cells were identified as melanocyte by morphological observation, Dopa staining and immunochemical staining.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2015年第12期1984-1990,共7页
Chinese Journal of Veterinary Science
基金
国家自然科学基金资助项目(31060330)
江西省教育厅科技资助项目(GJJ12221)
关键词
泰和乌骨鸡
黑色素细胞
鸡胚
体外培养
鉴定
Taihe silky fowls
melanocytes
chicken embryo
in vitro cultivation
identification
