Cell SELEX技术筛选巨噬细胞源性泡沫细胞适配子的初步研究

目的利用Cell SELEX技术筛选巨噬细胞源性泡沫细胞适配子。方法通过80mg/L氧化性低密度脂蛋白诱导THP-1巨噬细胞为泡沫细胞;聚合酶链反应扩增文库;生物素-链霉亲和素磁珠分离法构建单链DNA文库;以巨噬细胞源性泡沫细胞为靶细胞,巨噬细胞和血管平滑肌细胞为反筛细胞,利用Cell SELEX技术筛选与巨噬细胞源性泡沫细胞结合的寡核苷酸序列,并对最后一轮筛选结果进行克隆测序。结果 PCR的退火温度对PCR产物的量有显著的影响,退火温度由65℃提高为72℃时的产物量明显增加。利用生物素-链霉亲和素磁珠分离法获得了纯度高、产量大的ssDNA文库;经过18轮筛选后,成功获得了28个与预期大小相同的ssDNA序列。结论利用Cell SELEX技术筛选出了巨噬细胞源性泡沫细胞的适配子,为研发用于动脉粥样硬化的早期无创诊断试剂和靶向治疗药物奠定了基础。

Preliminary study on screening aptamers of macrophage-derived foam cells by Cell SELEX

Objective To obtain the aptamers of macrophage-derived foam cells by Cell SELEX.Methods THP-1 macrophages were induced into foam cells with 80 mg/L oxLDL;Random libraries were amplified by PCR;The technique basing on biotin-streptavidin-magnetism bead was applied to build a ssDNA library;Positive screening targing foam cells and negative screening targing macrophages and VSMC were alternated in Cell SELEX process.After the final round screening,the ssDNA were amplified by PCR with non-labeled primers,and then cloned and sequenced.Results PCR annealing temperature had a significant impact on PCR product.Amount of PCR product significantly increased when the temperature was from 65 ℃ to 72 ℃.More ssDNA with high purity were acquired by Biotin-streptavidin-magnetism bead separation method;After 18 rounds of screening,we successfully obtained 28 oligonucleotide sequences with the expected size.Conclusion We have acquierd aptamers of macrophage-derived foam cells by Cell SELEX,which will lay the foundation for developing early non-invasive diagnostic reagents and targeting therapies on AS.