基因编码钙指示剂在药物反应性细胞核钙信号实时动态检测中的应用

doi:10.3969/j.issn.1006-7795.2018.03.013

首都医科大学学报 ›› 2018, Vol. 39 ›› Issue (3): 378-384.doi: 10.3969/j.issn.1006-7795.2018.03.013

基因编码钙指示剂在药物反应性细胞核钙信号实时动态检测中的应用

龚明涛¹, 张晨光^1,2, 丁卫^1,2

1. 首都医科大学基础医学院生物化学与分子生物学系, 北京 100069;
2. 肿瘤侵袭和转移机制研究北京市重点实验室, 首都医科大学肿瘤研究所, 北京 100069

收稿日期:2017-05-15 出版日期:2018-05-21 发布日期:2018-06-11
通讯作者: 丁卫 E-mail:weiding@ccmu.edu.cn
基金资助:
国家自然科学基金（81372284）。

Genetically-encoded calcium indicator for detecting the dynamics of drug-induced nuclear calcium signals in real time

Gong Mingtao¹, Zhang Chenguang^1,2, Ding Wei^1,2

1. Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Capital Medical University, Beijing 100069, China;
2. Beijing Key Laboratory for Researches in Cancer Invasion and Metastasis, Cancer Institute of Capital Medical University, Beijing 100069, China

Received:2017-05-15 Online:2018-05-21 Published:2018-06-11
Supported by:
This study was supported by National Natural Science Foundation of China (81372284).

摘要/Abstract

摘要： 目的探讨新型基因编码钙指示剂（genetically-encoded calcium indicators，GeCIs）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GeCI变体——GCaMP6s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tdTomato融合作为分子内的定量参照。将构建的质粒分别转染至HeLa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。

关键词: 钙指示剂, 实时荧光成像, 核钙信号, 青蒿素, 高内涵

Abstract: 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 tdTomato with red fluorescence as an intramolecular reference to a most commonly used GeCI variant GCaMP6s, and tagged the molecule with the nucleoplasmin nuclear localization sequence (NLS) into an eukaryotic expression cassette. By transfecting HeLa or U87MG 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.

Key words: calcium indicator, live fluorescent imaging, nuclear signal, artemisinin, high-content

中图分类号:

龚明涛, 张晨光, 丁卫. 基因编码钙指示剂在药物反应性细胞核钙信号实时动态检测中的应用[J]. 首都医科大学学报, 2018, 39(3): 378-384.

Gong Mingtao, Zhang Chenguang, Ding Wei. Genetically-encoded calcium indicator for detecting the dynamics of drug-induced nuclear calcium signals in real time[J]. Journal of Capital Medical University, 2018, 39(3): 378-384.

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基因编码钙指示剂在药物反应性细胞核钙信号实时动态检测中的应用

Genetically-encoded calcium indicator for detecting the dynamics of drug-induced nuclear calcium signals in real time

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