基于核酸适体L4的转移性结直肠癌的靶向成像

Targeted Imaging of Metastatic Colorectal Cancer Based on Aptamer L4

摘要

研究了核酸适体L4的靶向结合特性，并探讨了其作为分子探针对临床结直肠癌患者组织的靶向成像能力，旨在为转移性结直肠癌的靶向诊断提供潜在的分子工具.采用流式细胞术分析了核酸适体L4的结合特异性、亲和性、温度稳定性和细胞选择性；通过酶处理实验探讨了核酸适体L4结合靶向分子的性质；基于生物素-链霉亲和素的结合原理，将核酸适体L4与量子点(Quantum dots，QDs)偶联形成了L4-QD探针，对临床结直肠癌患者组织进行了靶向成像，并分析了组织靶向性及与患者病理特征的相关性.结果表明，核酸适体L4可高特异性和高亲和力地结合靶细胞HCT116，解离常数(K_d)值为(10.4±1.7) nmol/L，且具有良好的温度稳定性.临床组织成像结果显示，L4-QD探针能够对结直肠癌组织实现靶向成像，且荧光强度与患者美国癌症联合委员会(American joint committee on cancer，AJCC)临床分期和生存预后不良呈显著正相关.核酸适体L4对转移性结直肠癌细胞具有高特异性和高亲和力，与量子点偶联可实现结直肠癌组织的靶向成像，有望成为评估结直肠癌进展程度的潜在分子探针及转移性结直肠癌靶向诊断的新型工具.

Abstract

To investigate the targeting and binding characteristics of aptamer L4 and evaluate its potential as a molecular probe for targeted imaging of colorectal cancer tissues in clinical samples, this study aimed to provide a novel tool for targeted diagnosis of metastatic colorectal cancer. Flow cytometry was employed to assess the binding specificity, affinity, thermal stability and cellular selectivity of aptamer L4. Enzymatic digestion assays were performed to determine the biochemical nature of the target molecule recognized by aptamer L4. Based on the biotin-streptavidin interaction, aptamer L4 was conjugated with quantum dots (QDs) to construct L4-QD probes, which were subsequently applied for targeted imaging of colorectal cancer tissues from clinical patients. The targeting specificity of the probes and their correlations with clinicopathological features were further analyzed. Aptamer L4 exhibited strong and specific binding to HCT116 cells, with a dissociation constant (K _d, nmol/L) of (10.4±1.7) nmol/L, and demonstrated favorable thermal stability. Aptamer L4 preferentially bound to tumor cells with metastatic potential, indicating that its target is likely a membrane-associated protein. Imaging of clinical specimens showed that L4-QD probes enabled specific visualization of colorectal cancer tissues, and the fluorescence intensity was significantly positively correlated with American joint committee on cancer (AJCC) stage and poor patient prognosis. In conclusion, aptamer L4 displays high specificity and affinity for metastatic colorectal cancer cells. Its conjugation with quantum dots allows effective targeted imaging of colorectal cancer tissues, highlighting its potential as a molecular probe for evaluating disease progression and as a novel tool for the targeted diagnosis of metastatic colorectal cancer.

关键词

转移性结直肠癌 / 核酸适体 / 靶向成像 / 量子点

Key words

Metastatic colorectal cancer / Aptamer / Targeted imaging / Quantum dots

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基金资助

国家自然科学基金(82373445)

辽宁省科学技术计划项目(2023JH2/20200155)