摘要
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-eGFP-LC3 （ptfLC3） 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-eGFP-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 trans- fected with plasmids, green spot increased in rapamycin treated HEK293 cells; while for mRFP-eGFP- LC3 plasmids transfeetion, green and red spot increased in rapamycin treated HEK293 cells. Conclu- sion： Dual-fluorescence mRFP-eGFP-LC3 is superior to mono-fluorescence GFP-LC3, which can com- prehensively reflect the level of autophagy and changes of autophagie flux, providing a reliable method for the quantitative analysis of autophagy.
出处
《贵阳医学院学报》
CAS
2015年第10期1029-1032,共4页
Journal of Guiyang Medical College
基金
国家自然科学基金青年项目(81300151)
