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橘色双冠丽鱼黑色素细胞和黄色素细胞的分离、培养与鉴定
宋红梅,周康奇,田雪,汪学杰,刘超,刘奕,牟希东,胡隐昌
1.中国水产科学研究院珠江水产研究所 农业农村部休闲渔业重点实验室 广东省现代休闲渔业工程技术研究中心 广东 广州 510380;2.淡水鱼类资源与生殖发育教育部重点实验室 重庆 402460;3.河南师范大学水产学院 河南省水产动物养殖工程技术研究中心 河南 新乡 453007
摘要:
为探讨橘色双冠丽鱼(Amphilophus citrinellus)黑色素细胞和黄色素细胞的分离、培养与鉴定方法,观察鱼类色素细胞生长特征,本研究以橘色双冠丽鱼的尾鳍、鳞片、皮肤组织为材料,在25℃~28℃条件下,分别在胰蛋白酶溶液和胶原酶溶液中消化6 h和12 h,可有效分离出色素细胞团,细胞悬液经气孔尼龙网过滤后,于35%-45%-55% Percoll试剂中梯度分离和收集黄色素细胞和黑色素细胞。采用K-SFM培养基进行细胞原代培养和细胞纯化,抑制成纤维细胞和角朊细胞的生长,用十四烷酰佛波醇乙酸酯(TPA)、双抗、bFGF的DMEM培养基进行色素细胞传代培养;运用巴染色和透射电镜观察细胞形态,并用分子标记技术进行细胞鉴定。结果显示,细胞分离时,黑色素细胞位于Percoll试剂45%和35%浓度层之间,黄色素细胞位于Percoll试剂35%浓度层上,2种色素细胞均凝聚成絮状。透射电子显微镜观察发现,黑色素细胞内部含大量黑色素小体,而黄色素细胞内部含喋呤体和类胡萝卜素囊泡;黑色素细胞左旋多巴(L-DOPA)染色呈阳性;取第10代色素细胞进行体色相关基因黑皮素1受体(melanocortin receptor 1, MC1R)、酪氨酸酶(tyrosinase, TYR)和牛内皮素受体B(endothelin receptor B, EDNRB) PCR检测,显示基因扩增条带特异性强,表明获得的2种色素细胞具有良好的生物学活性。本研究建立了橘色双冠丽鱼黄色素细胞和黑色素细胞的分离培养与鉴定方法,为进一步开展鱼类体色细胞分化和体色形成的分子机理研究提供了细胞模型。
关键词:  橘色双冠丽鱼  色素细胞  分离  培养  鉴定
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Isolation, culture and identification of polychromatic midas cichlids (Amphilophus citrinellus) melanophores and xanthophores
SONG Hongmei1,2,3, ZHOU Kangqi4, TIAN Xue5, WANG Xuejie1,2,3, LIU Chao1,2,3, LIU Yi1,2,3, MU Xidong1,2,3, HU Yinchang1,2,3
1.Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences;2.Key Laboratory of Leisure Fisheries, Ministry of Agriculture and Rural Affairs;3.Guangdong Modern Leisure Fisheries Engineering Technology Research Center, Guangzhou, Guangdong 510380, China;4.China Southwest University, Key Laboratory of Freshwater Fish Resources and Reproductive Development, Ministry of the Ministry of Education, Chongqing 402460, China;5.College of Fisheries, Engineering Technology Research Center of Henan Province for Aquatic Animal Cultivation, Henan Normal University, Xinxiang, Henan 453007, China
Abstract:
This study aimed to investigate the methods of isolation, culture, and identification of melanophores and xanthophores in polychromatic midas cichlids (Amphilophus citrinellus) and to thus observe the growth characteristics of pigment cells in fish. In this study, the caudal fin, scales, and skin tissues of A. citrinellus were used as the experimental material. To separate the pigment cells, the tissues were digested in trypsin solution and collagenase solution for 6 h and 12 h, respectively, at 25℃~28℃. After the cell suspension was filtered through a stoma nylon net, xanthophores and melanocytes were separated and collected by means of 35%-45%-55% Percoll multilayer density gradient centrifugation. K-SFM medium was used for primary cell culture and cell purification to inhibit the growth of fibroblasts and keratinocytes. DMEM supplemented with phorbol ester (TPA), double resistance, and bEGF, etc., was used for the subculture of pigment cells. The morphology of the cells was observed by L-DOPA staining and transmission electron microscopy, and the cells were identified by molecular markers. The results showed that when the cells were separated, the melanocytes were located at the boundary of the 45% and 35% Percoll reagent layers, while xanthophores were located on top of the 35% Percoll layer. Both pigment cells condensed into flocculation. Transmission electron microscopy showed that the melanocytes contained a large number of melanosomes, while the yellow cells contained the MTX and carotenoid vesicles. The melanocytes were positive under L-DOPA staining. The 10th generation of pigment cells was used for PCR detection of the body color related genes MC1R, TYR, and EDNRB, which showed strong specificity of gene amplification bands, indicating that the two pigment cells obtained had good biological activity. In this study, methods for the isolation, culture, and identification of melanophores and xanthophores of polychromatic midas cichlids were successfully established, providing a cell model for further research on the molecular mechanisms of body color cell differentiation and body color formation in fish.
Key words:  Amphilophus citrinellus  Pigment cell  Separation  Cultivation  Identification