中文摘要：

目的：筛选识别急性髓系白血病M2型（acute myeloblastic leukemia M2 subtype,AML-M2）CD33＋/CD34-细胞的核酸适体（aptamers）。方法：以AML-M2型白血病CD33＋/CD34-细胞为靶细胞,正常人CD33＋/CD34-细胞为反筛选细胞,采用细胞-指数富集配体系统进化（cell-systematic evolution of ligands by exponential enrichment,C-SELEX）技术,体外反复筛选单链DNA（single strand deoxyribonucleic acid,ssDNA）文库的适体,经聚合酶链式反应（polymerasechain reaction,PCR）扩增产生次级文库。然后将最终轮次的次级ssDNA文库克隆、测序并分析各适体的一级和二级结构。结果：琼脂糖凝胶电泳结果显示：每轮筛选出来的次级ssDNA文库PCR扩增产物均可见到清晰的DNA条带。经过13轮的反复筛选,次级ssDNA文库与细胞结合的荧光强度从2.14%上升到51.12%,至第13轮趋于稳定状态。对30个克隆子序列测定结果显示：22个适体一级结构分别含有AAGTA,TATCT,AGATG和AAATT 4个保守序列,另8个适体无此保守序列。二级结构模拟结果显示：30个适体含有4种不同的茎环、凸环模拟结构。结论：C-SELEX技术可用于急性髓系白血病原代细胞适体的筛选,筛选到的AML-M2型白血病CD33＋/CD34-细胞适体有望用于白血病的诊断与治疗。

英文摘要：

Objective： To screen aptamers binding CD33＋/CD34– cells from patients with acute myeloblastic leukemia M2 subtype（AML-M2）.Methods： CD33＋/CD34– cells from patients with AML-M2 were taken as targeted cells,CD33＋/CD34– cells from normal people were taken as anti-selecting cells,and aptamers in the single strand deoxyribonucleic acid（ssDNA） library were then selected repeatedly by cell-systematic evolution of ligands by exponential enrichment（C-SELEX） technology,and amplified by polymerase chain reaction（PCR） to generate sub-ssDNA library.During the experiment,PCR amplification with fluorescently labeled primer and flow cytometry were performed to analyze the aptamers’ enrichment of sub-library,and the final round product of the sub-ssDNA library was cloned.After the sequencing,the primary and secondary structures of the aptamers were analyzed. Results： Electrophoresis indicated that the product of PCR amplification for each round sub-ssDNA library was able to see a clear DNA band in the agarose gel.After 13 rounds of screening,the fluorescence intensity of the sub-ssDNA library binding the cells ranged from 2.14% to 51.12%,reaching a steady state at the 13th round.A total of 30 clones were selected and sequenced,22 of which contained 1 of the 4 conserved sequences of AAGTA,TATCT,AGATG and AAATT in their primary structure,but the remained eight aptamers contained none of the conserved sequence.Secondary structure analysis indicated that four stem-loops and loop simulation convex structures existed in the aptamers. Conclusion： C-SELEX technology can be used to screen the aptamers binding primary cells from patients with leukemia.The aptamers selected from the CD33＋/CD34– cells from the patients of AML-M2 subtype might be used for the diagnosis and treatment for leukemia.