摘要
原位、高时空分辨地检测细胞内氧化还原代谢状态是生命科学研究的一个瓶颈问题和迫切需求.然而,依赖细胞裂解、酶学、色谱、质谱等传统生化分析方法难以实时监测细胞内氧化还原代谢变化,更难以应用于高通量药物筛选.基于荧光蛋白的探针成像是近年来生命科学和医学领域迅速发展的一种分析检测技术.由于这些荧光探针实现了在活细胞内实时、动态地监测生物学过程,从而革命性地改变了生命科学研究.相对于化学小分子荧光探针,遗传编码的荧光蛋白探针在精确定位亚细胞结构、消除人为干扰以及活体应用方面,存在显著的优势.近年来,科学家针对细胞内重要的氧化还原代谢物,发明了多种多样的遗传编码荧光探针,实现了在单细胞、亚细胞甚至活体内对氧化还原代谢状态的特异性检测和成像,大大推动了相关研究领域的发展.本文将以细胞内两对关键的氧化还原代谢分子NADH/NAD^+和NADPH/NADP^+为例,重点介绍相关荧光探针的设计、性质、应用以及使用注意事项,以方便研究者更好地了解和使用相关技术.
In-situ measurement of intracellular redox metabolism with high spatiotemporal resolution remains to be an obstacle in life science research. However, traditional methods including enzymatic assays, chromatography and mass spectrometry require cell lysis, therefore suffer from some defects in real-time detection ofredox metabolism in living cells and are not suitable for high-throughput screen. Recently fluorescent imaging has become a powerful tool for biomedical research with the development of various genetically encoded fluorescent sensors as well as chemical probes. These approaches allow us to dynamically monitor biological processes in living cells, and have made revolutionary changes in life science research. Comparing with chemical probes, genetically encoded fluorescent sensors possess many advantages, including precise subcellular localization, minor artificial disturbance and high suitability for in vivo study. In the past decade, different types of genetically encoded fluorescent sensors have been developed to monitor redox metabolites, enabling imaging redox states at single-cell or sub-cellular resolution as well as in vivo and achieving exciting progresses in the research field ofredox biology. In this review, taking NADH/NAD＋ and NADPH/NADP＋ biosensors for example, we will give a detailed description on the design, property, application and practical concerns of genetically encoded fluorescent sensors to better understand and apply these tools.
作者
赵玉政
张卓
杨弋
ZHAO YuZheng ZHANG Zhuo YANG Yi(Synthetic Biology and Bioteehnology Laboratory, Optogeneties ＆ Molecular lmaging Interdisciplinary Research Center, East China University of Science and Technology, Shanghai 200237, China State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China)
出处
《中国科学：生命科学》
CSCD
北大核心
2017年第5期508-521,共14页
Scientia Sinica(Vitae)
基金
国家重点基础研究发展计划(批准号:2013CB531200)
国家自然科学基金(批准号:91313301,31225008,31470833,91649123,31671484)
上海市科委项目(批准号:14XD1401400,16430723100,15YF1402600)
111计划(批准号:B07023)
中国科协“青年人才托举工程”
上海市青年拔尖人才
高等学校博士学科点专项科研基金
生物反应器工程国家重点实验室和中央高校基本科研业务费资助
