军事医学2025，Vol.49Issue(4)：257-264,8.DOI:10.7644/j.issn.1674-9960.2025.04.003
ATP探针Chemo-G慢病毒载体的构建及其稳定转染细胞系的建立
Construction of an ATP probe Chemo-G lentiviral vector and establish-ment of a stable transfection cell line
摘要
Abstract
Objective To establish models for real-time dynamic monitoring of intracellular cytoplasmic adenosine triphosphate(ATP)and mitochondrial ATP levels in cells in order to study the changes in metabolic processes.Methods The lentiviral plasmids of the cytoplasmic chemogenetic green fluorescent protein(GFP)ATP probe(Chemo-G)and those of the mitochondrial-localized chemogenetic GFP ATP probe(mito-Chemo-G)were constructed before being transfected into HEK293T together with the helper plasmids,respectively.Chemo-G and mito-Chemo-G lentiviruses were obtained.HeLa cells were infected with the lentivirus.Puromycin resistance selection and flow cytometry cell sorting were employed to identify and isolate the infected HeLa cells.The growth and GFP expressions of HeLa cells were observed.A live-cell imaging system was used for continuous imaging of the cells,with stimuli added at specific time points to alter intracellular ATP levels to observe changes in the fluorescence intensity of the ATP probe.Results Lentiviral plasmids containing Chemo-G and mito-Chemo-G sequences were constructed.Two cell lines which could stably express Chemo-G and mito-Chemo-G were established that exhibited strong growth and accurate intracellular fluorescence localization.Live-cell imaging revealed that after the addition of 2-deoxy-D-glucose(2-DG)into HeLa-Chemo-G,the fluorescence resonance energy transfer(FRET)/GFP ratio showed a decrease that was partially reversed by the addition of glucose.The FRET/GFP ratio increased after histamine stimulation,but rapidly decreased after the addition of oligomycin.Conclusion The Chemo-G and mito-Chemo-G lentiviral vectors and stably transfected cell lines HeLa-Chemo-G and HeLa-mito-Chemo-G are constructed,which provides reliable experimental models for studying cellular metabolism and changes in intracellular ATP levels.关键词
三磷酸腺苷(ATP)/胞浆ATP探针/线粒体ATP探针Key words
adenosine triphosphate(ATP)/cytoplasmic ATP probe/mitochondrial ATP probe分类
化学引用本文复制引用
吴文俊,赵海鑫,高俊,王凯,韩秋影,李腾,潘欣..ATP探针Chemo-G慢病毒载体的构建及其稳定转染细胞系的建立[J].军事医学,2025,49(4):257-264,8.基金项目
国家杰出青年科学基金项目(82025028) （82025028）
