核酸适配子探针对转移性大肠癌细胞的多靶点成像及其靶标的定量分析

doi:10.7503/cjcu20160254

摘要/Abstract

摘要：

利用核酸适配子对肿瘤细胞的高亲和力靶向识别功能以及量子点的高荧光发射强度和光稳定性等特性, 制备了识别不同靶点的核酸适配子探针, 将其联合使用实现了对肿瘤细胞的多靶点成像及其靶标的定量分析. 使用通过Cell-SELEX 技术筛选得到的可特异性识别转移性大肠癌细胞系LoVo的7个核酸适配子, 分别与量子点QD605偶联制备分子探针. 基于流式细胞术的竞争实验结果表明, 7个探针可特异性识别靶细胞的不同靶点, 相互之间无识别干扰. 对靶细胞的荧光成像表明, 与单一探针相比, 多个探针联合使用可明显提高细胞表面的荧光信号强度, 且阳性细胞检出率明显增多, 显示出更高的检测灵敏度. 使用流式细胞术及荧光成像定量方法分析了7个探针对不同转移特性大肠癌细胞系的识别能力, 结果表明, 多个探针联合使用可有效评价大肠癌细胞的转移潜能. 本研究证实通过多个核酸适配子探针的联合使用可有效提高对靶细胞识别的灵敏度和准确性, 为核酸适配子的广泛应用及大肠癌的靶向诊断提供了新的思路和手段.

关键词: 核酸适配子, 转移性大肠癌, 多靶点, 量子点, 靶向成像

Abstract:

We developed seven aptamer probes towards metastatic colorectal cancer cells by conjugating the aptamers to quantum dots(QD605) for multi-targeted cancer cell imaging. The results of competitive assay showed that the seven aptamers could recognize their individual targets on the same cell without any mutual interference, revealing that they may be applied in combination for multi-target cancer cell imaging. The results of quantitative assay of the fluorescence images showed that compared with a single probe, the seven probes in combination can significantly increase the fluorescent intensity on the cell surface, and the detection rate of positive cells increased significantly, resulting in a higher detection sensitivity. Further, the probes were used to detect several colorectal cancer cell lines using flow cytometry. The results showed that seven probes used in combination could effectively evaluate colorectal cancer metastatic potential. This study demonstrated that usage of multiple aptamer probes in combination could effectively improve the sensitivity and accuracy of target imaging which provides a novel approach for targeted diagnosis of colorectal cancer.

Key words: Aptamer, Metastatic colorectal cancer, Multi-target, Quantum dot, Molecular imaging

中图分类号:

O657.3

TrendMD:

李婉明, 方瑾. 核酸适配子探针对转移性大肠癌细胞的多靶点成像及其靶标的定量分析. 高等学校化学学报, 2016, 37(7): 1262.

LI Wanming, FANG Jin. Quantitative Multi-targeted Imaging of Metastatic Colorectal Cancer Cells Using Aptamer Probes in Combination^†. Chem. J. Chinese Universities, 2016, 37(7): 1262.

图/表 8

Table 1 DNA sequences used in experiments

DNA probe	Sequence(5-3)
Library	AGGAGCAGCGTGGAGGATA(45N)TTAGGGTGTGTCGTCGTGGT
W1	ATTGATGACACTGAATTGATTTCAGGCATTGTTTGCTGCCACTTAGGGTGTGTCGTCG
W3	AGCAGCGTGGAGGATAGGGGTCGGAGTGGGTGGTTATGATTGGCTCTTCTGCGCTGC
W6	ACCCTATCGGTTTCTTGTCTTGCAAGCCTTCAGCTTCAGCAGCCTTAGGG
W9	ACGCAACCTCTTGGCTCATTGACTGAGGCCATTGTTTGTTGGCTTAGGGTGTG
W14	AGGATACAGGTTGGCTTTGGTGGCTATGGGTTGGGTGTGGTGCGTATCCT
W17	AGGAGCAGCGTGGAGGATATGATCCGACCGTTTTCGTTTGTTATGAACGGTGCTCC
W22	ACAGCGTGGAGGATATTGGTGTTGGTGGGCGTTTACGCTCCTTCGTCGCTG

Fig.1 Flow cytometry for the binding of FITC-labeled aptamers to LoVo(A1—A7) and HCT-8(B1—B7) cellsa. Library; b. aptamer. (A1, B1) W1; (A2, B2) W3; (A3, B3) W6; (A4, B4) W9; (A5, B5) W14; (A6, B6) W17; (A7, B7) W22.

Fig.2 Quantitative results based on flow cytometry Data were presented as the mean±standard deviation.

Fig.3 Mutual effects of aptamers on the recognition of targets by competitive experiment(A) FITC-W1; (B) FITC-W3; (C) FITC-W6; (D) FITC-W9; (E) FITC-W14; (F) FITC-W17; (G) FITC-W22.

Fig.4 Fluorescence(A1—A9) and optical(B1—B9) images of LoVo cells stained by aptamers-QDAll pictures were taken under a fluorescence microscope with × 200 magnification.(A1, B1) Library; (A2, B2) W1; (A3, B3) W3; (A4, B4) W6; (A5, B5) W9; (A6, B6) W14; (A7, B7) W17; (A8, B8) W22; (A9, B9) multiple aptamers.

Fig.5 Quantitative results based on QD-cellsa. Library; b. W1; c. W3; d. W6; e. W9; f. W14; g. W17; h. W22; i. multiple aptamers.

Fig.6 Flow cytometry assays of the binding of aptamers with cells (A) LoVo; (B) HCT116; (C) SW620; (D) CL187; (E) HEK293.

Fig.7 Relative fluorescence intensities of aptamers-cells

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