贵州医科大学学报

2015, v.40;No.181(10) 1029-1032

[打印本页] [关闭]
本期目录(Current Issue) | 过刊浏览(Past Issue) | 高级检索(Advanced Search)

双荧光mRFP-eGFP-LC3体系在细胞自噬中的作用
Evaluating the Effect of Dual-Fluorescence mRFP-e GFP-LC3 in Autophagy

黄桢钧;刘彬;刘本荣;刘少军;
HUANG Zhenjun;LIU Bin;LIU Benrong;LIU Shaojun;Department of Cardiology,Guangzhou Institute of Cardiovascular Disease,the Second Affiliated Hospital of Guangzhou Medical University;

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;Department of Cardiology,Guangzhou Institute of Cardiovascular Disease,the Second Affiliated Hospital of Guangzhou Medical University;

Email:

DOI: 10.19367/j.cnki.1000-2707.2015.10.007

文章评论(Comment):

序号(No.) 时间(Time) 反馈人(User) 邮箱(Email) 标题(Title) 内容(Content)
反馈人(User) 邮箱地址(Email)
反馈标题(Title)
反馈内容(Content)
扩展功能
本文信息
服务与反馈
本文关键词相关文章
本文作者相关文章
中国知网
分享