在国家自然科学基金等的资助下，湖南大学化学生物传感与计量学国家重点实验室王柯敏教授课题组在核酸适配体的肿瘤活体荧光分子成像研究中，首次提出了基于细胞膜蛋白触发构型变化的“激活式核酸适配体探针”概念，设计合成了一种针对肿瘤细胞特异性表达蛋白的发夹型激活式核酸适配体探针，显著提高肿瘤细胞成像反差、缩短检测时间，成功用于裸鼠肿瘤活体实时荧光成像。相关研究结果发表在美国科学院院刊上（PNAS 2011, 108, 3900-3905）。自然出版集团著名期刊SciBX用核酸适配体成像（Imaging with aptamers）为题对该工作迅速以封面故事（Cover Story）形式进行了亮点报道和评述。
核酸适配体（又称Aptamer）是利用核苷酸之间严格的识别能力和亲和力而设计的人工合成寡核苷酸，并通过指数富集配体的系统进化（SELEX）技术筛选而获得。它不仅具有类似抗体对靶标高特异性和高亲和力的特点，更在许多方面优于抗体，如：靶标种类丰富（包括离子、有机染料、蛋白、完整的肿瘤细胞等）、合成方法简便且重复性好、修饰灵活以及便于长期贮存和常温运输等。特别是作为一种分子量小、阴离子性且无免疫原性的分子探针，核酸适配体在活体内具有组织渗透和吞噬速率快，血液和非靶组织滞留时间短和靶组织聚集效率高等优势，是一种潜在理想的活体成像探针。传统的核酸适配体活体成像探针主要是采用“always on”模式，由于信号始终存在，在活体内往往表现出背景信号高、检测时间长、成像对比度不高和灵敏度有限等缺点。如果能够巧妙设计只有在特定靶肿瘤目标刺激下才能产生可检测信号的激活式核酸适配体成像探针，则有可能显著降低活体成像背景，缩短检测时间，提高肿瘤成像对比度和检测灵敏度，为活体肿瘤的诊断分析提供一种更为理想的成像模式。
针对活体肿瘤的高灵敏度、高特异性检测需要，该课题组创新性的提出了“激活式核酸适配体探针”的概念，以人类急性白血病细胞CCRF-CEM细胞的核酸适配体为模型，构建了针对CCRF-CEM细胞膜表面肿瘤标志性蛋白的激活式核酸适配体探针，不仅成功实现了缓冲液和血清体系中CCRF-CEM细胞的快速、灵敏、特异检测以及活体内CCRF-CEM肿瘤的高对比度和特异性诊断成像，而且与传统“always on”单荧光标记核酸适配体探针相比，成像反差显著提高，检测时间也由原来的数小时缩短至十几分钟。该研究不仅为核酸适配体在肿瘤活细胞检测和活体成像研究中的应用提供了全新的手段和思路，而且为肿瘤活体荧光分子成像领域开发了一类具有普遍适用潜力的激活式分子探针，具有重要的科学价值和广泛的临床前实验以及临床实验应用前景。（生物谷Bioon.com）
生物谷推荐原文出处：
PNAS doi: 10.1073/pnas.1016197108
Activatable aptamer probe for contrast-enhanced in vivo cancer imaging based on cell membrane protein-triggered conformation alteration
Hui Shi, Xiaoxiao He, Kemin Wang1, Xu Wu, Xiaosheng Ye, Qiuping Guo, Weihong Tan, Zhihe Qing, Xiaohai Yang, and Bing Zhou
Aptamers have emerged as promising molecular probes for in vivo cancer imaging, but the reported “always-on” aptamer probes remain problematic because of high background and limited contrast. To address this problem, we designed an activatable aptamer probe (AAP) targeting membrane proteins of living cancer cells and achieved contrast-enhanced cancer visualization inside mice. The AAP displayed a quenched fluorescence in its free state and underwent a conformational alteration upon binding to target cancer cells with an activated fluorescence. As proof of concept, in vitro analysis and in vivo imaging of CCRF-CEM cancer cells were performed by using the specific aptamer, sgc8, as a demonstration. It was confirmed that the AAP could be specifically activated by target cancer cells with a dramatic fluorescence enhancement and exhibit improved sensitivity for CCRF-CEM cell analysis with the cell number of 118 detected in 200 μl binding buffer. In vivo studies demonstrated that activated fluorescence signals were obviously achieved in the CCRF-CEM tumor sites in mice. Compared to always-on aptamer probes, the AAP could substantially minimize the background signal originating from nontarget tissues, thus resulting in significantly enhanced image contrast and shortened diagnosis time to 15 min. Furthermore, because of the specific affinity of sgc8 to target cancer cells, the AAP also showed desirable specificity in differentiating CCRF-CEM tumors from Ramos tumors and nontumor areas. The design concept can be widely adapted to other cancer cell-specific aptamer probes for in vivo molecular imaging of cancer.
