黄颡鱼黑色素细胞原代培养及迁移相关基因克隆分析

Primary Culturing of Melanoma Cells and Cloning Analysis of Migrationrelated Gene in Pelteobagrus fulvidraco

黑素皮质素受体-1(MC1R)在鱼体中对黑色素细胞的分化及迁移起主要的调控作用,而且在神经系统和免疫系统中均表现出重要的生理功能。本研究对黄颡鱼表皮黑色素细胞的培养条件进行探索,并在此基础上对MC1R基因进行克隆,为后续研究黄颡鱼黑色素异常的细胞及分子机理研究提供基础。结果表明,黄颡鱼表皮黑色素细胞在27℃培养条件下,72 h开始贴壁生长,培养基中添加20%小牛血清培养效果比添加10%小牛血清好。此外,我们对黄颡鱼MC1R基因进行克隆。MC1R基因克隆方面,黄颡鱼MC1R基因全长为936 bp,编码312个氨基酸,与剑尾鱼(Xiphophorus maculates)、孔雀鱼(Poecilia reticulata)、条斑星鲽(Verasper moseri)、海鲈(Dicentrarchus labrax)、大菱鲆(Psetta maxima)等5种鱼类MC1R基因的同源性分别为97%、96%、90%、90%、90%,系统分析结果显示,黄颡鱼MC1R基因在进化树上的位置与黄颡鱼的分类所处位置基本吻合。黄颡鱼表皮黑色素细胞原代培养的条件为:27℃,添加20%小牛血清,所克隆基因为黄颡鱼MC1R基因。本研究为后续研究黄颡鱼黑色素异常的细胞及分子机理研究提供基础。

Melanocortin receptor-1 （MC1R）played a major role in the regulation of cell differentiation and mi-gration in melanoma cells, and also had an important physiological function in the nervous system and the im-mune system in fish. In this study, we explored the culture conditions of epidermal melanocytes in Pel-teobagrus fulvidraco. The results showed that Pelteobagrus fulvidraco epidermal melanocytes adhered atter 72h at 27 -12. The culture medium supplemented with 20% fetal calf serum was preferred compared to 10% sup-plemented during the culture. In addition, we cloned 936-bp MC1R gene which is encoding 312 amino acids inPelteobagrus fulvidraco. The homologies between Pelteobagrusfulvidraco and other fishes including swordtailfish （Xiphophorus maculates）, guppy （Poecilia reticulata）, streak Star flounder （Verasper moseri）, sea bass （Di-centrarchus labrax） and turbot （Psetta maxima） were 97%, 96%, 90%, 90% and 90%, respectively. System a-nalysis showed that Pelteobagrus fulvidraco MC1R gene in the position of evolutionary tree was coincidewith the location of classification. Consensus results in Pelteobagrus fulvidraco were observed based on mo-lecular evolutionary analysis on MC1R gene and taxonomy. It was proved that the primary culture conditionsof Pelteobagrus fulvidraco epidermal melanocytes in is ： 27℃, supplemented with 20% fetal calf serum, and thecloned gene was Pelteobagrus fulvidraco MC1R. Our research could provide a basis for subsequent resear-ches on the cellular and molecular mechanism of lacking melanin symptoms in Pelteobagrus fulvidraco.