中文摘要：

基因表达谱显示,绵羊角蛋白2（keratin2,Krt2）的mRNA在不同毛色皮肤的表达不同,暗示Krt2基因可能对皮肤黑色素生成有一定的影响。为探索角蛋白2对体外培养的羊驼皮肤黑色素细胞黑色素生成的影响,首先采用PCR扩增产物测序,结合DNAMAN软件比对分析发现,羊驼Krt2编码序列（c DNA）与NCBI公布的人KRT2高度同源（91%）;将人KRT2添加于培养的羊驼皮肤黑色素细胞,观察人KRT2对羊驼皮肤黑色素细胞黑色素的生成作用。免疫组织化学显示,外源性的人KRT2处理羊驼皮肤黑色素细胞72 h后,黑色素细胞的细胞质中Krt2表达增强。实时定量PCR及Western印迹实验揭示,与胎牛血清清蛋白处理的羊驼皮肤黑色素细胞比较,1 ng/m L、10 ng/m L和100 ng/m L人KTR2处理的羊驼皮肤黑色素细胞酪氨酸酶（Tyr）、酪氨酸相关蛋白-1（Tyrp1）、小眼畸形相关转录因子（Mitf）基因表达明显上调（P〈0.05）;尤其在添加10 ng/m L KTR2的细胞中,3个基因的mRNA相对表达水平升高尤其显著,分别是对照细胞的4倍、10倍和12.9倍（P〈0.01）,蛋白质相对表达水平分别是对照细胞的2倍、2.1倍和1.7倍（P〈0.01）。分光光度法测量A490结果证明,1 ng/m L、10 ng/m L和100 ng/m L人KTR2处理的羊驼皮肤黑色素细胞产生的黑色素含量分别是对照细胞的1.3倍（P〈0.05）、1.8倍（P〈0.01）和1.5倍（P〈0.05）。上述结果说明,人KTR2处理可通过刺激羊驼皮肤黑色素细胞黑色素合成相关信号通路,促进黑色素的合成。

英文摘要：

The gene expression profile shows that keratin2（ Krt2） gene is differently transcribed in sheep skins with different coat colors,implying that Krt2 may be involved in the production of melanin.To investigate the effect of Krt2 on the melanogenesis of alpaca skin melanocytes,alpaca Krt2-coding sequence（ c DNA） was obtained by RT-PCR and sequenced,followed by the alignment of nucleotide sequences of Krt2 c DNAs from several animals and human. Comparison of alpaca Krt2-coding sequence with that of human KRT2 revealed 91% identity. Hence,the human KRT2 protein was used to treat the alpaca skin melanocytes in culture to examine its role in melanogenesis. Immunohistochemistry studyshowed that exogenous human KRT2 was localized mainly in the cytoplasm in cultured alpaca skin melanocytes. Real-time quantitative RT-PCR（ RT-q PCR） and Western blotting analysis revealed that compared with the melanocytes exposed to fetal bovine serum（ FBS; control）,exposure to KTR2 with the concentrations of 1,10 and 100 ng / m L led to up-regulation of both mRNA and protein levels of tyrosinase（ Tyr）,tyrosinase-related protein 1（ Tyrp1） and microphthalmia-associated transcription factor（ Mitf）genes in alpaca skin melanocytes（ P 0. 05）; particularly in the melanocytes exposed to 10 ng / m L KRT2,the mRNA levels of these three genes were 4-,10- and 12. 9-folds of those in BSA-exposed melanocytes（ P 0. 01）,and the levels of these proteins were 2-,2. 1- and 1. 7-folds of those in FBSexposed melanocytes（ P 0. 01）. Furthermore,spectrum analysis showed that produced melanin levels in melanocytes exposed to KRT2（ 1,10 and 100 ng / m L） were significantly increased by 1. 3-,1. 8- and1. 5-folds,when compared with FBS-exposed（ control） cells（ P 0. 05,P 0. 01 and P 0. 05,respectively）. These data suggest that human KRT2 and alpaca Krt2 may promote melanogenesis in alpaca skin melanocytes through stimulating melanin- formation signals in the alpaca skin melanocytes.