黄桢钧,刘彬,刘本荣,刘少军
HUANG Zhenjun,LIU Bin,LIU Benrong,LIU Shaojun

• 1:广州心血管疾病研究所广州医科大学附属第二医院心内科

摘要(Abstract)：

目的:探讨双荧光mRFP-e GFP-LC3(ptf LC3)体系在细胞自噬中的作用。方法:采用不同浓度雷帕霉素(50、100、200、500、1 000 nmol/L)处理人胚肾细胞(HEK293),蛋白印迹法检测不同浓度雷帕霉素组中自噬标记蛋白(LC3蛋白)的表达及LC3-II/LC3-I比值;单荧光GFP-LC3质粒和双荧光mRFP-e GFP-LC3质粒转染HEK293细胞,雷帕霉素(200 nmol/L)处理3 h,采用荧光显微镜统计,并比较两种方法转染后HEK293细胞内LC3亮点的变化。结果:不同浓度雷帕霉素处理HEK293细胞,LC3-II蛋白和LC3-II/LC3-I比值均增加;转染GFP-LC3质粒时,雷帕霉素处理HEK293细胞中绿色亮点的数量增加;转染mRFP-e GFP-LC3质粒时,雷帕霉素处理HEK293细胞中绿色和红色亮点数量均增加。结论:双荧光mRFP-e GFP-LC3体系优于单荧光GFP-LC3体系,更能全面完整地反映细胞自噬水平,能够反映细胞内自噬流的变化,是自噬定量分析的可靠方法。
Objective: To investigate the effect of dual-fluorescence mRFP-e GFP-LC3( ptf LC3) in autophagy. Methods: HEK293 cells were treated with rapamycin( 50,100,200,500,1 000 nmol / L)for 3h and then adopting Western blot to test the expression of LC3 protein and ratio of LC3-II / LC3-I.Mono-fluorescence GFP-LC3 and dual-fluorescence mRFP-e GFP-LC3 transfected HEK293 cells. 24 h after the transfection,the cells were treated with rapamycin( 200 nmol / L) for an additional 3 h,then analyzed by fluorescence microscopy to compare the spot change of LC3 in HEK293 cells. Results:Different concentration of rapamycin increased the ratio of LC3-II / LC3-I in HEK293 cells. After transfected with plasmids,green spot increased in rapamycin treated HEK293 cells; while for mRFP-e GFPLC3 plasmids transfection,green and red spot increased in rapamycin treated HEK293 cells. Conclusion: Dual-fluorescence mRFP-e GFP-LC3 is superior to mono-fluorescence GFP-LC3,which can comprehensively reflect the level of autophagy and changes of autophagic flux,providing a reliable method for the quantitative analysis of autophagy.

关键词(KeyWords)： 雷帕霉素;自噬;人胚肾细胞;LC3;单荧光GFP-LC3质粒;双荧光mRFP-e GFP-LC3
rapamycin;autophagy;human embryonic kidney cells;LC3;mono-fluorescence GFP-LC3 plasmid;dual-fluorescence mRFP-e GFP-LC3

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金青年项目(81300151)

作者(Author): 黄桢钧,刘彬,刘本荣,刘少军
HUANG Zhenjun,LIU Bin,LIU Benrong,LIU Shaojun

DOI: 10.19367/j.cnki.1000-2707.2015.10.007

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