高表达Snail蛋白诱导黑色素瘤EMT细胞模型的构建及鉴定  

Construction and Verification of an Inducible EMT Model in Mouse Melanoma Stably Overexpressing Snail

作　　者：方瑞[1] 郭强[1] 杜军[1] 

机构地区：[1]中山大学药学院,广州510006

出　　处：《中国生物工程杂志》2013年第7期1-7,共7页China Biotechnology

基　　金：国家"973"计划(2011CB935800);国家自然科学基金(81272311;81071712)资助项目

摘　　要：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 establish an inducible EMT cell model which stably overexpressing recombinant mouse Snail and evaluate its application in fluorescence imaging in vitro and in vivo.Methods: The mouse Snail gene was amplified from pCMV6-mSnail by PCR and cloned into eukaryotic expression plasmid pL-tdTomato-Neo.The recombinant colonies were identified by double enzyme digestion and DNA sequencing.The mouse melanoma B16 cells were transfected with recombinant plasmid pL-tdTomato-mSnail and the stably transfected cells B16/Td-mSN were selected with culture media supplemented with G418.The mRNA and protein levels of Snail as well as EMT markers E-cadherin and Vimentin were examined by qPCR and Western blot.Also,the B16 and B16/Td-mSN cells were inoculated subcutaneously into the nude mice respectively to prepare allografted tumor model and examined by fluorescence imaging system.Results: The recombinant expression plasmid pL-tdTomato-mSnail was constructed correctly,in which the red fluorescence protein tdTomato,mouse Snail and resistance gene were seriesly connected by two 2A peptides.The selected B16/dT-mSN cells express high levels of Snail and red fluorescence protein.Also,the cells display elongated and spindle-like morphology as well as down-regulation of E-cadherin and up-regulation of Vimentin.The allograft mouse model for in vivo imaging was successfully established by use of this stable cell line.And the fluorescence signal was stable and strong.Conclusion: An EMT cell model stably expressing recombinant mouse Snail and red fluorescence protein was successfully established.The cell model provides a convenient,intuitive and stable tool for imaging studies of Snail biological function during tumor EMT progress.

关 键 词：稳定细胞株 SNAIL 黑色素瘤 上皮-间质转化 荧光成像 

分 类 号：R739.5[医药卫生—肿瘤]