Zhu Ping, Wang Guangping, Zhang Shuqin, Xu Yajing, Peng Minyuan, Yin Hui, Chen Yan, Tan Sanqin, Chen Fangping
Department of Hematology, Xiangya Hospital, Central South University, Changsha 410008, China.
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2012 Aug;37(8):771-6. doi: 10.3969/j.issn.1672-7347.2012.08.003.
To screen aptamers binding CD33+/CD34- cells from patients with acute myeloblastic leukemia M2 subtype (AML-M2).
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.
Electrophoresis indicated that the product of PCR amplification for each round subssDNA 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.
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.
从急性髓细胞白血病M2亚型（AML-M2）患者中筛选与CD33+/CD34-细胞结合的适配体。
将AML-M2患者的CD33+/CD34-细胞作为靶细胞，正常人的CD33+/CD34-细胞作为反选细胞，然后通过指数富集配体的细胞系统进化（C-SELEX）技术对单链脱氧核糖核酸（ssDNA）文库中的适配体进行反复筛选，并通过聚合酶链反应（PCR）扩增以生成亚ssDNA文库。实验过程中，采用荧光标记引物的PCR扩增和流式细胞术分析亚文库适配体的富集情况，并对亚ssDNA文库的终轮产物进行克隆。测序后，分析适配体的一级和二级结构。
电泳显示各轮亚ssDNA文库PCR扩增产物在琼脂糖凝胶中可见清晰的DNA条带。经过连续13轮筛选，与细胞结合亚ssDNA文库的荧光强度在2.14%至51.12%之间，第13轮达到稳定状态。共挑选30个克隆进行测序，其中22个在其一级结构中包含AAGTA、TATCT、AGATG和AAATT这4个保守序列中的1个，但其余8个适配体均不包含保守序列。二级结构分析表明，适配体中存在4个茎环和环模拟凸起结构。
C-SELEX技术可用于筛选与白血病患者原代细胞结合的适配体。从AML-M2亚型患者的CD33+/CD34-细胞中筛选出的适配体可能用于白血病的诊断和治疗。
