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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。
Currently, little is known about surface-specific molecular markers of acute leukemia cells, and the diagnosis of leukemia is still lacking in specific methods for leukemia cells. In this study, we used a cell-systematic evolution of ligands by exponentiation (cSELEX) technique to screen for AML-M2 patients with acute myeloblastic leukemia (AML) CD33 + / CD34 + cell-associated aptamers provide the basis for finding surface-specific molecular markers for AML-M2 leukemia cells. Firstly, the bone marrow CD33 + / CD34 + cells of AML-M2 patients were sorted by immunomagnetic beads method and used as target cells. Normal human CD33 + / CD34 + cells were used as anti-screening cells. Single strand deoxyribonucleic acid , ssDNA) libraries were screened for aptamers that bind to AML-M2CD33 + / CD34 + cells. Subsequently, the structure of each aptamer was analyzed by cloning and sequencing. The results showed that after 13 rounds of screening, the enrichment of ssDNA library aptamer and CD33 + / CD34 + cells in AML-M2 increased from 0.7% to 52.9%, and stabilized to the 13th round. Analysis of the 30 aptamers obtained showed that most aptamers contain one of the CCCCT, CTCTC and CTCAC conserved sequences. Secondary structure analysis revealed that 30 aptamers contained three different types of simulated secondary structures. Conclusion: The aptamer of CD33 + / CD34 + cells from AML-M2 leukemia cells was successfully screened in this study. This provided the basis for further molecular finding of surface-specific molecular markers of AML-M2 leukemia cells and AML-M2 leukemia.