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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。
Objective: To screen for aptamers that recognize acute myeloblastic leukemia M2 subtype (AML-M2) CD33 + / CD34- cells. Methods: AML-M2 leukemia CD33 + / CD34- cells as target cells, normal human CD33 + / CD34- cells as anti-screening cells, using cell-systematic evolution of ligands by exponential enrichment C-SELEX) was used to screen the aptamer of single strand deoxyribonucleic acid (ssDNA) library in vitro. The secondary library was amplified by polymerase chain reaction (PCR). The final round of secondary ssDNA libraries are then cloned, sequenced and analyzed for the primary and secondary structures of each aptamer. Results: The results of agarose gel electrophoresis showed that a clear DNA band could be seen in the PCR amplification products of secondary ssDNA library screened each round. After 13 rounds of repeated screening, the fluorescence intensity of secondary ssDNA library bound to cells increased from 2.14% to 51.12%, and stabilized to the 13th round. The results of 30 clones showed that the 22 aptamers contained four conserved sequences of AAGTA, TATCT, AGATG and AAATT, respectively, and the other eight aptamers did not. The secondary structure simulation results show that: 30 aptamers contain four different stem and loop analog structures. Conclusion: The C-SELEX technique can be used to screen primary aptamers of acute myeloid leukemia. The selected AML-M2 leukemia CD33 + / CD34- cell aptamers are expected to be used in the diagnosis and treatment of leukemia.