泡沫细胞靶向适配子的体外筛选

Screening Aptamers of Foam Cells Derived From Macrophages by Complex SELEX

目的筛选巨噬细胞源性泡沫细胞的寡核苷酸适配子,为动脉粥样硬化的靶向治疗提供实验依据。方法以80 mg/L氧化型低密度脂蛋白孵育THP-1巨噬细胞72 h,建立泡沫细胞模型;利用指数富集配基的系统进化技术,从体外合成的随机单链DNA文库中筛选特异性寡核苷酸适配子;荧光显微镜观察寡核苷酸文库与泡沫细胞结合的特异性;克隆、测序确定适配子的序列并进行一级结构和二级结构分析。结果通过油红O染色和高效液相色谱分析,确定成功建立泡沫细胞模型;经过18轮的循环筛选,寡核苷酸文库仅与泡沫细胞结合,不再结合巨噬细胞、血管平滑肌细胞。测序鉴定出的所有适配子序列可以分为12个家族,没有共同的同源序列。二级结构分析表明,适配子主要形成茎环结构,这可能是适配子与巨噬细胞源性泡沫细胞结合的结构基础。结论利用复合靶指数富集配基的系统进化技术成功筛选出巨噬细胞源性泡沫细胞的寡核苷酸适配子。

Aim To acquire oligonucleotide aptamers of foam cells derived from macrophages and laying theoretical basis for targeted therapy of atherosclerosis. Methods THP-1 cell was treated with 80 mg/L oxidized low density lipoprotein（ox-LDL） for 72 hours to establish macrophage derived foam cell model.Aptamers acquired from a ssDNA library by complex system evolution of ligands by exponential enrichment（SELEX）.Fluorescence microscopy was used to detect the binding specificity of ssDNA library and foam cells.After cloning and sequencing,the primary sequences and second structure of the aptamers were analyzed. Results THP-1 macrophage derived foam cell model was identified successfully by red oil O staining and HPLC.After 18 rounds selection,the ssDNA library did not bind to THP-1 macrophages and vascular smooth cells but bind to foam cells.All aptamers had no conserved motifs,and could be divided into 12 families.The main secondary structures of these aptamers were stem-loops which could be vital in the interaction between aptamers and foam cells. Conclusions We obtained the aptamers of THP-1 macrophage derived foam cells successfully by complex SELEX.