一种体内实时观察黑色素瘤的斑马鱼模型

摘要

目的 建立一种在体内实时观察黑色素瘤发生发展的斑马鱼模型,为黑色素瘤的相关研究提供一个便捷的活体模型.方法 构建红色荧光蛋白标记的黑色素瘤表达系统pMITFa1-RedV12/hRas,利用小鼠B16黑色素细胞验证其表达效果.再将pMITFa1-RedV12/hRas载体导入1-细胞期的斑马鱼受精卵中,采用活体成像系统实时观察黑色素瘤在斑马鱼体内的形成过程.结果 pMITFa1-RedV12/h Ras载体在B16细胞中成功表达.显微注射pMITFa1-RedV12/hRas载体到1-cell期的斑马鱼受精卵3 d后,在荧光显微镜下观察到荧光标记的黑色素瘤;8周后,肉眼可以明显观察到黑色素瘤.黑色瘤的成瘤率达100％.pMITFa1-RedV12/h Ras载体被显微注射到TG (zlyz:EGFP) 转基因斑马鱼的受精卵中后,在活体成像系统下清晰地观察到红色荧光标记的黑色素瘤细胞与绿色荧光标记的巨噬细胞的相互作用.结论 成功构建了经济简便、易操作的在体内实时观察黑色素瘤的斑马鱼模型.%Objective To establish a zebrafish model for real-time monotoring the development of melanoma in vivo and screening relative drugs. Methods The expression effect of pMITFa1-RedV12/h Ras plasmid, in which the melanoma was labeled by red fluorescent protein (RFP), was verified in mouse B16 melanocytes. After microinjecting pMITFa1-RedV12/h Ras into 1-cell stage of fertilized eggs of zebrafish, we monitored the process of melanoma formation with in vivo imaging system in zebrafish.Results pMITFa1-RedV12/h Ras was successfully expressed in mouse melanoma B16 cells. Three days after microinjection of pMITFa1-RedV12/h Ras vector to 1-cell satge of zebrafish, melanomas labeled by RFP protein was observed under the fluorescence microscope. Eight weeks later, melanomas were observed clearly by eyes. The rate of tumor formation was 100％. When microinjected into pMITFa1-RedV12/h Ras vector into fertilized eggs of TG (zlyz:EGFP) transgenic zebrafish, the interaction between the melanoma cells labeled by RFP protein and the macrophages labeled by green fluorescent protein was clearly observed in in vivo imaging system. Conclusion Successfully established a zebrafish model for real-time monitoring melanoma in vivo, which is cheap and easy to operate.