文章摘要
橘色双冠丽鱼体表黑色素细胞和黄色素细胞的分离、培养与鉴定
Isolation, Culture and Identification of Polychromatic Midas cichlids (Amphilophus citrinellus) Melanophores and Xanthophores
投稿时间:2020-06-17  修订日期:2020-08-20
DOI:
中文关键词: 橘色双冠丽鱼  色素细胞  分离  培养  鉴定
英文关键词: Amphilophus citrinellus  pigment cell  Separation  cultivation  identification
基金项目:
作者单位E-mail
宋红梅 中国水产科学研究院珠江水产研究所 shm1227@126.com 
周康奇 淡水鱼类资源与生殖发育教育部重点实验  
田雪 河南师范大学水产学院  
汪学杰 中国水产科学研究院珠江水产研究所  
刘超 中国水产科学研究院珠江水产研究所  
刘奕 中国水产科学研究院珠江水产研究所  
牟希东 中国水产科学研究院珠江水产研究所  
胡隐昌① 中国水产科学研究院珠江水产研究所  
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中文摘要:
      为探讨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6h和12h,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,用添加TPA、双抗、bEGF等的DMEM培养基进行色素细胞的传代培养;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percell试剂45%和35%浓度层之间,黄色素细胞位于percell试剂35%浓度层上,两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有大量黑色素小体,而黄色素细胞内部含喋呤体和类胡萝卜素囊泡;黑色素细胞进行L-Dopa染色呈阳性;取第10代色素细胞进行体色相关基因MC1R、TYR和EDNRB的PCR检测,显示基因扩增条带特异性强,表明获得的两种色素细胞具有良好的生物学活性。本研究成功建立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细胞模型。
英文摘要:
      In order to study the characteristics of fish skin pigment cells, methods for isolating , culturing and identification melanophores and xanthophores from caudal fin, scales and skin tissue of Polychromatic Midas cichlids (Amphilophus citrinellus) were harvested and digested in trypsin and collagenase solution respectively at first time and secondary. The mixed cell suspension from stomatal nylon mesh filter were filtered from 35% - 45% - 55% of percoll gradient reagent. Then, the melanophores and xanthophores were collected respectively. Primary cell culture and l purification were carried out by K-SFM medium which inhibited the growth of fibroblasts and keratinocytes, and wereScarried out in DMEM medium. In addition, TPA, double antibody and bEGF were added to culture pigment cells. Cell morphology was observed by electron microscopy and electron microscopy, and the cells were identified by molecular markers. The results showed that the melanocytes were located between 45% and 35% of percell reagent, while the xanthophores were located at the 35% concentration of percell reagent, and the two separated pigment cells were condensed into flocculent. Transmission electron microscopy (tem) showed that a large number of melanosomes are present in the in melanocytes, and methotrexate and carotenoid vesicles lived in xanthophores. The third generation of melanocytes were positive for l-dopa staining. Two kinds of pigment cells in the 10th generation were detected by PCR using skin color related genes MC1R, TYR and EDNRB, which showed that the bands of gene amplification were specific, indicating that the obtained melanin and yellow pigment cells had good biological activity. In this study, a method for isolation, culture and identification of melanophores and xanthophores in Polychromatic Midas cichlids was successfully established for the first time, and the culture conditions of the cells were optimized to provide a cell model for the research on the molecular mechanism of fish body color cell differentiation and body color formation. In this study, a method for isolation, culture and identification of melanocytes and xanthoxanthin in orange biloba cichlid was successfully established for the first time, and the culture conditions of the cells were optimized to provide a cell model for the research on the molecular mechanism of fish body color cell differentiation and body color formation.
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