摘要
目的探讨新型基因编码钙指示剂(genetically-encoded calcium indicators,Ge CIs)在药物反应性细胞核钙信号实时动态检测中的应用。方法将常用的Ge CI变体——GCa MP6s用核定位序列(nuclear localization sequence,NLS)标记以用于细胞核钙的检测;同时在其C端与红色荧光蛋白td Tomato融合作为分子内的定量参照。将构建的质粒分别转染至He La细胞和U87MG细胞中,利用活细胞共聚焦显微成像技术对核钙信号的实时变化进行动态监测。结果蕈青霉素(Paxilline)能够引起在40 min连续记录中核钙水平的持续增加,并且增加的动力学模式在不同的细胞系中差异存在统计学意义;同时在药物处理后细胞反应的筛选中显示,青蒿素可以引起细胞核钙浓度的明显改变,其变化的幅度与持续情况与蕈青霉素及过氧化氢的作用有所不同。此外,笔者还发现利用核钙的变化指标可以对细胞在接受应激性刺激后的异质性表现进行统计聚类分析。结论在非兴奋细胞中,不同药物刺激可以引起细胞核钙离子浓度的升高。
Objective To explore the application of genetically-encoded calcium indicator for detecting the dynamics of drug-induced nuclear calcium signals in real time. Methods We fused td Tomato with red fluorescence as an intramolecular reference to a most commonly used Ge CI variant GCa MP6 s,and tagged the molecule with the nucleoplasmin nuclear localization sequence（ NLS） into an eukaryotic expression cassette. By transfecting He La or U87 MG cells,and imaged with a live confocal microscope,we were able to quantitatively monitor the dynamic changes of nuclear calcium signals in real time. Results We found that the ability of Paxilline to increase nuclear calcium concentrations varied among different cell types. We also found that various chemical,including artemisinin and several cell stress inducing reagent,were able to elevate nuclear calcium level in a relatively persistent manner. Conclusion Different drugs can increase calcium concentration in non-excitable cells.
作者
龚明涛
张晨光
丁卫
Gong Mingtao;Zhang Chenguang;Ding Wei(Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China;Beijing Key Laboratory for Researches in Cancer Invasion and Metastasis, Cancer Institute of Capital Medical University, Beijing 100069, China)
出处
《首都医科大学学报》
CAS
北大核心
2018年第3期378-384,共7页
Journal of Capital Medical University
基金
国家自然科学基金(81372284)~~
关键词
钙指示剂
实时荧光成像
核钙信号
青蒿素
高内涵
calcium indicator
live fluorescent imaging
nuclear signal
artemisinin
high-content
