文章摘要
庄 燕,贲晶晶,柏 惠,陈 琪.稳定表达GFP-LC3的RAW264.7细胞系的建立[J].南京医科大学学报,2009,29(6):757~761766
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
投稿时间:2009-01-06  
DOI:10.7655
中文关键词: GFP-LC3  基因转染  RAW264.7细胞  稳定表达  自噬
英文关键词: RAW264.7 cell  GFP-LC3  gene transfection  stable expression  autophagy
基金项目:国家重点基础研究发展计划(973计划)资助项目(2006CB708509)
作者单位
庄 燕 南京医科大学动脉粥样硬化研究中心,江苏 南京 210029 
贲晶晶  
柏 惠  
陈 琪  
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中文摘要:
      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?
英文摘要:
      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 transfection 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 confocal microscope and Western blot. Results:Selected by G418,2 transfected 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.
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