动脉粥样硬化靶向适配子的亲和力筛选

Affinity screening of targeting aptamers for atherosclerosis

目的：筛选动脉粥样硬化高亲和力适配子,为动脉粥样硬化靶向引导物的体内应用提供实验基础。方法将前期研究获得的一组巨噬细胞源性泡沫细胞适配子中的12个适配子进行生物素标记,采用酶联寡聚核苷酸吸附试验（ELONA）方法测定其亲和力（Kd）,采用高脂方法建立载脂蛋白E基因敲除（ApoE-/-）动脉粥样硬化模型,将亲合力最高的适配子Cy5进行荧光标记,激光共聚焦显微镜下观察其与动脉粥样硬化病变组织的结合能力。结果12个巨噬细胞源性泡沫细胞的适配子均具有较好的亲和力,解离平衡常数（Kd值）范围在4.33-39.58 nmol/L之间,其中9号适配子（Apt9）的亲和力最高,Kd值为（4.33±0.33）nmol/L,体内亲和力检测发现Apt9能特异性结合ApoE-/-动脉粥样硬化血管斑块病变组织。结论筛选的Apt9适配子能与巨噬细胞源性泡沫细胞和动脉粥样硬化病变血管组织特异性结合,可以用于进一步的体内研究。

Objective To screen high affinity aptamers for atherosclerosis, so as to provide experimental basis for the application in vivo of targeting leading materials of atherosclerosis. Methods Twelve aptamers of a group of aptamers in macrophage derived foam cells achieved from preliminary study were made biotin labelling, the enzyme-linked oligonu-cleotide assay （ELONA） was used to detect its affinity （Kd）, the high-fat feeding was used to establish ApoE-/-atherosclero-sis model, the highest affinity of aptamer Cy5 was made fluorescence labeling, then the combining capacity of it with atherosclerotic pathologic tissues was observed under the laser scanning confocal microscope. Results The aptamers of 12 macrophage derived foam cells all had good affinity. The range of equilibrium dissociation constant （Kd） was 4.33-39.58 nmol/L, of which, the affinity of Apt9 was the highest, the Kd value was （4.33±0.33） nmol/L, the detection of affinity in v iv o showed that Apt9 could be specifically bound to ApoE-/-atherosclerotic pathologic tissues. Conclusion The screened aptamer Apt9 can be specifically bound to macrophage derived foam cells and atherosclerotic pathologic tissues, which can be used for further study in vivo.