急性髓系白血病CD33^+/CD34^+细胞表面核酸适配体的筛选及结构分析  

Screening and Structure Analysis of Nucleic Acid Aptamers Binding to Surface of CD33^+/CD34^+ Cells from Patients with Acute Myeloid Leukemia Subtype M_2

作　　者：张书芹[1,2] 王光平[1] 朱平[1] 梁嘉佳[1] 徐雅静[1] 彭敏源[1] 陈炎[1] 谭三勤[1] 陈方平[1] 

机构地区：[1]中南大学湘雅医院血液科,湖南长沙410008 [2]北京市道培医院

出　　处：《中国实验血液学杂志》2011年第3期561-565,共5页Journal of Experimental Hematology

基　　金：国家自然科学基金资助项目;编号30871094

摘　　要：目前,对急性白血病细胞表面特异性分子标记物所知甚少,因而白血病的诊断尚缺乏白血病细胞的特异性方法。为此,本研究采用细胞-指数富集配体系统进化(cell-systematic evolution of ligands by exponentialenrichment,cSELEX)技术,筛选与急性髓系白血病(acute myeloblastic leukemia,AML)M2型(AML-M2)患者CD33+/CD34+细胞结合的核酸适配体(aptamer),为寻找AML-M2白血病细胞表面特异性分子标记物提供基础。首先,通过免疫磁珠方法分选出AML-M2患者骨髓CD33+/CD34+细胞并将其作为靶细胞,正常人CD33+/CD34+细胞为反筛选细胞,采用cSELEX技术,从单链DNA(single strand deoxyribonucleic acid,ssDNA)文库中筛选与AML-M2CD33+/CD34+细胞结合的适配体。随后,通过克隆和测序分析各适配体的结构。结果显示,经过13轮次的反复筛选,ssDNA文库的适配体与AML-M2患者CD33+/CD34+细胞的富集度从0.7%增加到52.9%,至第13轮时趋于稳定。对所获得的30个适配体序列分析表明,大多数适配体含有CCCCT、CTCTC和CTCAC保守序列中的一种。二级结构分析显示,30个适配体中含有3种不同类型的模拟二级结构。结论 :本研究成功筛选到AML-M2型白血病CD33+/CD34+细胞的适配体,这为进一步寻找AML-M2白血病细胞表面特异性分子标记物以及AML-M2型白血病的分子诊断创造了基础。A little is known about the specific marker on the surface of acute leukemia cells, leading to the lack of the specific diagnosis method for acute leukemia. Therefore, in this study, cell-systematic evolution of ligands by exponential enrichment （cSELEX） was performed to screen the aptamers binding to CD33 ＋/CD34＋ cells from the patients with acute myeloblastic leukemia （AML） of M2 subtype （AML-M2 ） so as to provide the basis for finding the specific marker on the surface of AML-M2 CD33 ＋/CD34 ＋ cells. Firstly, AML-M2 CD33 ＋/CD34＋ cells were sorted and used as targeted cells, and normal CD33 ＋/CD34＋ cells were used as counter-targeted cells ; the aptamers binding to CD33 ＋/CD34＋ cells from patients with AML-M2 were screened from the single strand deoxyribonucleic acid （ssDNA） library by cSELEX. Subsequently, each aptamer structure was analyzed after cloning and sequencing. The results indicated that after 13 round of screenings, the enrichment of aptamers in the ssDNA library was ranged from 0.7% to 52.9%, and reached steady state at 13th round screening. Sequence analysis for 30 aptamers showed that most of the aptamers born one of the three conserved sequences of CCCCT, CTCTC, and CTCAC. Secondary structure analysis indicated that three different secondary structures existed in these aptamers. It is concluded that the aptamers binding to the AML-M2 CD33 ＋/CD34 ＋ cells are successfully screened, which lay the basis for further looking for the specific marker on the surface of AML-M2 CD33 ＋/CD34 ＋ cells, and the molecular diagnosis of the AML-M2 leukemia.

关 键 词：细胞-指数富集配体系统进化技术 CD33+/CD34+细胞 急性髓系白血病 M2型 适配体 

分 类 号：R733.7[医药卫生—肿瘤]