稳定表达GFP-LC3的RAW264.7细胞系的建立  

Establishment of a stable GFP-LC3-expressed RAW264.7 cell line

作　　者：庄燕[1] 贲晶晶[1] 柏惠[1] 陈琪[1] 

机构地区：[1]南京医科大学动脉粥样硬化研究中心,江苏南京210029

出　　处：《南京医科大学学报（自然科学版）》2009年第6期757-761,766,共6页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家重点基础研究发展计划(973计划)资助项目(2006CB708509)

摘　　要：目的:建立稳定表达GFP-LC3的小鼠巨噬细胞RAW264.7细胞系。方法:构建pcDNA3.1-GFP-LC3真核表达载体,应用转染技术将该质粒导入RAW264.7细胞,用G418筛选稳定表达的细胞系。真核细胞中GFP-LC3的表达分别用荧光显微镜与Western blot方法检测,并利用该稳定表达细胞系观察内质网应激时细胞发生自噬的情况。结果:成功获得2株转染并经G418反复筛选的RAW264.7细胞系,在倒置荧光显微镜下观察可见绿色荧光的表达率在95%以上,Western blot结果证实了GFP-LC3融合蛋白的表达。激光共聚焦显微镜和Western blot均证明内质网应激可以诱导自噬的发生。结论:用pcDNA3.1-GFP-LC3转染的RAW264.7细胞经G418筛选,可成功建立GFP-LC3稳定表达系,从而为后续功能实验提供有用的细胞研究模型。Objective：To establish a stable GFP-LC3-expressed RAW264.7 cell line. Methods：The pcDNA3.1-GFP-LC3 plasmid was constructed and transfected into RAW264.7 cell with transfeetion reagent. The stable transfectants were screened by G418. The GFP-LC3 protein expression was analyzed by Western blot. The fluorescent signals were detected by inverted fluorescence microscope. ER stress-induced autophagy was detected by eonfocal microscope and Western blot. Results：Selected by G418,2 transfeeted cell lines showed high expression level of GFP-LC3 ,as demonstrated by Western blot analysis. More than 95% cells showed positive fluorescent signals under inverted fluorescence microscope. The formation of autophagosomes and the increases in the conversion of LC3- I to LC3-II was observed in the constructed cells when treated with the ER stress inducer,thapsigargin. Conclusion：A RAW264.7 cell line stably expressing GFP-LC3 was constructed successfully in the study.

关 键 词：GFP-LC3 基因转染 RAW264.7细胞 稳定表达 自噬 

分 类 号：R758.63[医药卫生—皮肤病学与性病学]