流式细胞术在检测细胞氧化还原状态荧光蛋白探针中的应用  

Flow cytometry detection of cellular redox status with genetically encoded fluorescent probe

作　　者：田烨[1] 刘奥 董瑞 祁星 易静[1] 杨洁[1] 

机构地区：[1]上海交通大学基础医学院生物化学与分子细胞生物学系,上海200025

出　　处：《上海交通大学学报（医学版）》2018年第1期10-17,共8页Journal of Shanghai Jiao tong University:Medical Science

基　　金：国家自然科学基金(31230037);上海市自然科学基金(16ZR1418400)~~

摘　　要：目的·探索流式细胞术检测基因编码的荧光蛋白探针ro GFP2的方法,比较其与经典的激光扫描共聚焦显微镜(laser scanning confocal microscope,LSCM)检测方法的异同,探讨其在检测不同氧化还原状态细胞亚群中的应用价值。方法·用ro GFP2转染He La细胞,经H_2O_2处理后,采用LSCM技术和流式细胞术分别检测ro GFP2,以405 nm/488 nm荧光信号强度比值反映细胞氧化还原状态。转染胰腺癌细胞株Panc-1细胞,应用流式细胞术检测CD44和CD24不同标记细胞亚群的ro GFP2,比较干细胞与其他细胞氧化还原状态的异同。结果·与LSCM技术相比,流式细胞术也可检测H_2O_2引起的ro GFP2变化,且可区分群体中不同的亚群。Panc-1细胞中CD24^+/CD44^+肿瘤干细胞群与非干细胞群相比,ro GFP2比值更低,细胞处于更还原的状态。结论·流式细胞术可用于检测基因编码的氧化还原探针和特异地分析亚群的变化,可能将应用于肿瘤生物学、干细胞生物学等研究中。Objective. To test the application of flow cytometry technique to detect the redox status with genetically encoded fluorescent probe roGFP2, to compare it with laser scanning confocal microscope （LSCM）, and to demonstrate the diversity of cellular redox status in HeLa and Pane-1 cell populations. Methods Time lapse imaging with LSCM was performed in single cell transfected with roGFP2 probe to detect the dynamic changes of 405 nm/488 nm ratio （405/488 ratio）. Flowcytometry technique was also performed in HeLa cells transfected with roGFP2 probe to detect the dynamic alterations of 405/488 ratio. The global cell population was analyzed and the subpopulations of different redox status were dissected. Flowcytometry technique was further applied in Panc-1 cells with different CD24 or CD44 marker to detect the dynamic alterations of 405/488 ratio of roGFP2 and identify the different redox status. Results. Time lapse imaging with LSCM showed that 405/488 ratio of roGFP2 dramatically changed in response to H2O2 in dose and time dependent manner at a single cell level. When dozens of cells were chosen, the average ratio showed increased and dynamic trend. Compared to LSCM, flow cytometry could detect the average of 405/488 ratio of roGFP2 as well. Meanwhile, with the application of flow cytometry the cell population can be divided into three subpopulations based on 405/488 ratios, most in oxidized and medium redox status, few in reduced status. Using flow cytometry, CD24^＋/ CD44^＋ Panc- 1 cells, pancreatic cancer stem cells, can be found to have overall lower 405/488 ratio and more percentage of subpopulation of reduced status under resting and stress condition. Conclusion, Flow cytometry technique can be applied to detect roGFP2 and has advantage in application to show the overall as well as diverse redox status in cell population. Flow cytometry detection of cellular redox status with genetically encoded probe can be a useful tool in tumor cell biology and developmental biology research.

关 键 词：流式细胞术 细胞氧化还原状态 基因编码荧光蛋白探针 roGFP2 肿瘤干细胞 

分 类 号：R34[医药卫生—基础医学]