Abstract: Objective　To establish a method for the isolation and identification of melanocytes from the skin of albino mutant chinese hamsters (AL) and wild-type hamsters (WT), and to analyze the functions of melanin synthesis and cell proliferation, in order to provide an ideal primary cell model for the study of albinism pathogenesis in this spontaneous albino mutant animal model.Methods　Hamster back skin tissues within 3 days of birth were removed, and melanocytes were isolated by trypsin digestion and cultured in MelM medium. Melanocytes were identified by immunofluorescence staining, L-Dopa staining, and transmission electron microscopy.The melanin content was determined by Masson-Fontana staining and colorimetric assay,the tyrosinase (TYR) activity was detected by L-Dopa catalytic assay and MTT assy, the cell proliferation was determined by cell cycle analysis and colony formation assy.Results Immunofluorescence of melanocyte-specific proteins TYR and Pmel17 showed that isolated AL and WT skin melanocytes all expressed. L-Dopa staining of AL melanocytes was significantly lighter than that of WT melanocytes, and there were fewer intracellular maturation-stage melanosomes. Melanin content and TYR activity of AL melanocytes were significantly lower than that of WT melanocytes (P<0.001); the proliferation rate of AL melanocytes was significantly faster than that of WT melanocytes (P<0.05), the proportion of S-phase cells was significantly higher (P<0.05), and the colony-forming ability was significantly enhanced (P<0.01).Conclusion　Trypsin digestion can obtain functional melanocytes from albino mutant chinese hamster skin, which have weakened melanin synthesis but enhanced proliferation ability.

Key words: Chinese hamster, melanocytes, isolation and culture

摘要： 目的　建立白化突变黑线仓鼠(AL)及野生型黑线仓鼠(WT)皮肤黑色素细胞分离鉴定方法,分析黑色素合 成功能及细胞增殖,为该自发白化突变动物模型白化致病机制研究提供理想的原代细胞模型。方法　取出生3 d 以内的仓鼠背部皮肤组织,胰蛋白酶消化法分离黑色素细胞,培养于MelM培养基;用免疫荧光染色、L-Dopa染色、 透射电镜等方法鉴定黑色素细胞,Masson-Fontana染色及比色法测定黑色素含量,L-Dopa催化法检测酪氨酸酶 (TYR)活性,MTT、细胞周期分析及克隆形成测定细胞增殖。结果　黑色素细胞特异性蛋白TYR、Pmel17免疫荧光 显示,分离的AL和WT皮肤黑色素细胞均表达;AL黑色素细胞L-Dopa染色明显浅于WT黑色素细胞,并且细胞内 成熟期黑色素小体较少;AL黑色素细胞黑色素含量及TYR活性显著低于WT黑色素细胞(P<0.001);AL黑色素 细胞增殖速度明显快于WT黑色素细胞(P<0.05),S期细胞比例显著升高(P<0.05),并且克隆形成能力明显增强 (P<0.01)。结论　胰蛋白酶消化法能获得性状稳定的白化突变黑线仓鼠皮肤黑色素细胞,该细胞黑色素合成减 弱,但增殖能力增强。

关键词: 黑线仓鼠, 黑色素细胞, 分离培养