| 张李钰,刘瑞萍,杨 颖,孙宏利,王 猛,武海滨.基于量子点荧光共振能量转移（FRET）效应的裂开型CD20核酸适配体探针用于非霍奇金淋巴瘤的检测研究[J].,2019,19(18):3432-3437 |
| 基于量子点荧光共振能量转移（FRET）效应的裂开型CD20核酸适配体探针用于非霍奇金淋巴瘤的检测研究 |
| Detection of NHL Cells based on Split CD20 Aptamer and QDs-mediated FRET Effect |
| 投稿时间：2019-01-28 修订日期：2019-02-23 |
| DOI：10.13241/j.cnki.pmb.2019.18.007 |
| 中文关键词: 核酸适配体 CD20 量子点 FRET 非霍奇金淋巴瘤 |
| 英文关键词: Aptamer CD20 Quantum dot FRET NHL |
| 基金项目:陕西省重点研发项目（2017-SF-280）；西安市科技计划项目（SF1510(4)） |
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| 中文摘要: |
| 摘要 目的：构建新型的基于量子点荧光共振能量转移效应的裂开型CD20核酸适配体激活式荧光探针用于非霍奇金淋巴瘤（Non-Hodgkin Lymphoma, NHL）的检测。方法：将CD20核酸适配体CE4-1进行裂解，结合量子点和Cy5荧光供受体队之间的FRET效应，利用流式细胞术，对裂开型核酸适配体的裂开位置、比例、浓度和反应时间进行优化，并在最优条件下评估该检测体系对CD20+细胞的检测特异性。结果：将CD20核酸适配体CE4-1分别裂开成CE4-1-1a/CE4-1-1b和CE4-1-2a/CE4-1-2b两种组合，分别修饰量子点（QD）或荧光受体Cy5后两两组合，分别与CD20 sup>+细胞孵育，流式细胞术发现CE4-1-1a/CE4-1-2b组合时信号最强；进一步，通过探索两种探针比例、探针浓度、孵育时间等参数，发现当CE4-1-1a和CE4-1-2b浓度比例为1:5、CE4-1-1a浓度为4 nM、孵育时间为50 min时，该裂开型核酸适配体体系显示出对靶肿瘤细胞最强的亲和力和FRET信号激活性能。进一步，实验发现该体系与CD20 sup>+细胞（Raji和Ramos）可产生较强信号，而与CD20－细胞（Jurkat、K562、Min6和Hela）均未产生阳性信号，提示该探针可有效保持对CD20 sup>+细胞的高特异性。结论：基于量子点荧光共振能量转移（FRET）效应的裂开型CD20核酸适配体探针体系在检测CD20 sup>+细胞方面表现出了极低的背景信号，同时有着较好的FRET信号值，有望实现CD20 sup>+ NHL细胞的高灵敏激活式检测。 |
| 英文摘要: |
| ABSTRACT Objective: To construct novel splited CD20 aptamer system based on FRET for the detection of NHL．Methods: CD20 aptamer CE4-1 was splited as CE4-1-1a/CE4-1-1b group or CE4-1-2a/CE4-1-2b group. Splited aptamers were modified with QD or Cy5 separately, and incubated with CD20+ cells. Cells were evaluated by flow cytometry and CE4-1-1a/CE4-1-2b group generated the strongest signal. Further, the ratio, concentration and incubation time of splited aptamer system were explored, and the detection specificity was ecaluated. Results: It is shown that when the ratio of CE4-1-1a/CE4-1-2b was 1:5, the concentration of CE4-1-1a was 4 nM, and the incubation time was 50 min, the splited aptamer system exhibited the strongest FRET signal. In addition, this system generated relative string signal with CD20+ cell lines (Raji and Ramos), whereas exhibited no positive signal with CD20－ cell lines (Jurkat, K562, Min6 and Hela), indicating the detection specificity to CD20+ cells. Conclusion: The CD20 splited aptamer system based on FRET for the detection of NHL exhibits weak background signal and relatively strong FRET signal, which presents a promising method for detection of CD20+ NHL cells. |
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