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
程炜轩;许国焕;熊达;张丽;吴清洋;郭莹姿;谭文俊;
摘要(Abstract):
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。
关键词(KeyWords): 黑素皮质素受体;原代培养;克隆;黄颡鱼
基金项目(Foundation): 广东省科学院青年科学研究基金
作者(Author): 程炜轩;许国焕;熊达;张丽;吴清洋;郭莹姿;谭文俊;
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DOI:
参考文献(References):
- [1]徐建瑜,刘鹰.基于计算机视觉的罗非鱼适应环境的体色变化研究[J].水生生物学报,2009,33(2):164-168Xu J Y,Liu Y.Study on colormodification of tilapia Oreochromis Niloticus under environmental background and stress by computer vision[J].Acta Hydrobiologica Sinica,2009,33(2):164-168(in Chinese)
- [2]Parichy D M.Animal pigment pattern:An integrative model system for studying the development,evolution,and regeneration of form[J].Seminars in Cell and Developmental Biology,2009,20:63-64
- [3]Andersson T P,Filippini D,Suska A.Frog melanophores cultured on fluorescent microbeads:Biomimic-based Biosensing[J].Biosensors and Bioelectronics,2005,21:111-120
- [4]Wolf K,Quimby M C.Established eurythermic line of fish cells in vitro[J].Science,1962,135(3508):1065-1066
- [5]Logan D W,Burn S F,Jackson I J.Regulation of pigmentation in zebrafish melanophores[J].Pigment Cell Research,2006,19(3):206-213
- [6]Jennifer R,Pia R L,Natalie L R,et al.Mc1r pathway regulation of zebrafish melanosome dispersion[J].Zebrafish,2008,5(4):289-295
- [7]张卓然.培养细胞学与细胞培养技术[M].上海:上海科学技术出版社,2004:23-57
- [8]孔祥婷,沈颂东,姜德勋,等.黄颡鱼吻端细胞培养研究[J].淡水渔业,2009,39(2):19-25Kong X T,Shen S D,Jiang D X,et al.A preliminary study on snout tissue culture from Pelteobagrus fulvidraco[J].Freshwater Fisheries,2009,39(2):19-25(in Chinese)
- [9]王武,余卫忠,石张东.江黄颡鱼适宜生长水温的研究[J].水产科技情报,2003,30(1):13-15Wang W,Yu W Z,Shi Z D.Effects of water temperature on growth of Pseudobagrus vachelli[J].Fisheries Science and Technology Information,2003,30(1):13-15(in Chinese)
- [10]Mcquillan D J,Handley C J,Campbell M A,et al.Stimulation of proteoglycan biosynthesis by serum and insulinlike growth factor-I in cultured bovine articular cartilage[J].Biochemical Journal,1986,240(2):423-430
- [11]Kobayashi Y,Chiba H,Mizusawa K,et al.Pigment-dispersing activities and cortisol-releasing activities of melanocortins and their receptors in Xanthophores and head kidneys of the goldfish Carassius auratus[J].General and Comparative Endocrinology,2011,173(3):438-446
- [12]Klovins J,Haitina T,Fridmanis D,et al.The melanocortin system in Fugu:Determination of POMC/AGRP/MCR gene repertoire and synteny,as well as pharmacology and anatomical distribution of the MCRs[J].Molecular Biology and Evolution,2004,21(3):563-579
- [13]Schi th H B,Haitina T,Ling M K,et al.Review evolutionary conservation of the structural,pharmacological,and genomic characteristics of the melanocortin receptor subtypes[J].Peptides,2005,26(10):1886-900
- [14]Haitina T,Klovins J,Takahashi A,et al.Functional characterization of two melanocortin(MC)receptors in lamprey showing orthology to the MC1 and MC4 receptor subtypes[J].BMC Evolutionary Biology,2007,7(1):101
- [15]Cieslak M,Reissmann M,Hofreiter M,et al.Colours of domestication[J].Biological Reviews,2011,86(4):885-899
- [16]Braasch I,Brunet F,Volff J N,et al.Pigmentation pathway evolution after whole-genome duplication in fish[J].Genome Biology and Evolution,2009,1(15):479-493
- [17]Selz Y,Braasch I,Hoffmann C,et al.Evolution of melanocortin receptors in teleost fish:The melanocortin type I receptor[J].Gene,2007,401(1-2):114-122
- [18]Van der Salm A L,Pavlidis M,Flik G,et al.The acute stress response of red porgy,Pagrus pagrus,kept on a red or white background[J].General and Comparative Endoerinology,2006,145(3):247-253
- [19]李捷.中国南方鲿科鱼类的动物地理与系统进化[D].广东:华南师范大学,2006:1-2
- [20]杨干荣.湖北鱼类志[M].湖北:科学技术出版社,1987:159