目的:构建稳定表达Snail蛋白的可用于肿瘤上皮-间质化研究的肿瘤细胞模型。方法:采用亚克隆方法从质粒pCMV6-mSnail中PCR扩增小鼠Snail基因,连接至表达质粒pL-tdTomato-Neo,筛选重组质粒并经双酶切及测序鉴定。构建成功的重组质粒pL-tdTomato-mSnail转染小鼠黑色素瘤B16细胞,G418筛选稳定细胞株。采用荧光定量PCR和Western blot技术检测胞内Snail及上皮/间质标志物的变化。建立裸小鼠皮下移植瘤模型,活体成像系统观测移植瘤。结果:重组质粒pL-tdTomato-mSnail成功构建,其稳定转染株B16/dT-mSN胞体发出强烈红色荧光。胞内Snaill水平显著上调,E-钙粘蛋白下调,波形蛋白表达上调,呈现典型的上皮-间质化表型。结论:成功获得稳定高表达小鼠Snail蛋白的EMT细胞模型,且可用于体内外荧光成像观测,为研究Snail蛋白在介导肿瘤EMT过程中的生物作用提供了重要的实验工具。 OBJECTIVE: To construct a tumor cell model which can be used for the study of tumor epithelial-interstitial cells stably expressing Snail protein. Methods: The mouse Snail gene was amplified by PCR from the plasmid pCMV6-mSnail and ligated into the expression plasmid pL-tdTomato-Neo. The recombinant plasmid was screened and identified by double enzyme digestion and sequencing. The constructed recombinant plasmid pL-tdTomato-mSnail was transfected into mouse melanoma B16 cells and G418 was used to screen stable cell lines. Fluorescent quantitative PCR and Western blot were used to detect the changes of intracellular Snail and epithelial / mesenchymal markers. A subcutaneous xenograft tumor model was established in nude mice and the in vivo imaging system was used to observe the xenografts. Results: The recombinant plasmid pL-tdTomato-mSnail was successfully constructed and its stable transfection cell line B16 / dT-mSN emitted strong red fluorescence. Significant upregulation of intracellular Snaill levels, E-cadherin downregulation, elevated vimentin, showing a typical epithelial-interstitial phenotype. CONCLUSION: The EMT cell model of stable and highly expressed mouse Snail protein was successfully obtained and could be used for fluorescence imaging observation in vitro and in vivo, which provided an important experimental tool for studying the biological role of Snail protein in mediating tumor EMT.