大鼠骨髓间充质干细胞特异性核酸适配子的筛选和鉴定

Selection and Identification of DNA Aptamers against Mesenchymal Stem Cells from Rat Bone Marrow

目的利用指数富集的配体系统进化技术(SELEX)筛选与大鼠骨髓间充质干细胞(BMSCs)特异性结合的核酸适配子,并对核酸适配子与大鼠BMSCs的结合方式及能力进行鉴定。方法利用SELEX技术,以分离获取的大鼠BMSCs为靶点,从构建的核酸文库中最大程度富集与BMSCs特异性结合的核酸序列,流式细胞术检测文库富集的特异程度,荧光共聚焦实验观察文库与BMSCs的结合能力。对富集程度最高的文库进行克隆、序列测定及序列分析,获得候选适配子。使用RNA structure软件对候选适配子的二级结构进行预测,流式细胞术测定不同浓度候选适配子与BMSCs结合后的平均荧光强度,使用SigmaPlot软件按公式Y=BmaxX/(X+Kd)获得每个候选适配子与BMSCs的结合曲线及平衡解离常数(Kd)。挑选与BMSCs结合力最高的适配子,流式细胞术检测其与不同来源BMSCs的结合能力。结果流式细胞术与荧光共聚焦实验表明SELEX筛选第9轮文库与BMSCs结合能力最高。经序列分析获得8个大鼠BMSCs特异性候选适配子,均能与BMSCs高亲和力结合,其中适配子apt7的亲和力最高,Kd值为(11.79±0.72)nmol/L。细胞选择性实验表明apt7仅与大鼠来源的BMSCs高亲和力结合,而不能结合小鼠及人来源的BMSCs。结论通过SELEX技术成功筛选出能高亲和力、高特异性结合大鼠BMSCs的适配子apt7,此方法为BMSCs的鉴定提供了新思路和方法,同时为适配子应用于组织工程支架表面招募循环干细胞提供了实验依据。

Objective To select specific aptamers from bone marrow mesenchymal stem cells(BMSCs)of rat,and to investigate the binding mode and capture ability of the aptamers with the BMSCs in vitro.Methods Systematic evolution of ligands by exponential enrichment(SELEX)technology was applied to obtain aptamers binding to BMSCs of rat.After the enrichment of the nucleic acid sequence specific binding to BMSCs,DNA pool was cloned and sequence analysis helped to obtain candidate aptamers.Flow cytometric analysis and confocal imaging showed the binding affinity between aptamers and BMSCs.Different concentrations of aptamers were incubated with BMSCs,then mean fluorescence intensity was calculated and applied to the formula Y=BmaxX/(X+Kd)to generate Kd by SigmaPlot software.The aptamers that had the best binding affinity was incubated with BMSCs from mouse or human.Results Flow cytometry and fluorescence confocal imaging showed that the library from the ninth round of SELEX screening had the highest binding ability to BMSCs.We obtained eight BMSCs-specific candidate aptamers by SELEX technology.Flow cytometry and confocal imaging showed these candidate aptamers could bind to BMSCs with high affinity.And apt7 showed the best affinity to BMSCs with Kd of(11.79±0.72)nmol/L.In addition,apt7 could only bind to BMSCs of rat while not with BMSCs from mouse or human.Conclusion In this study,specific aptamer(apt7)for BMSCs of rat was successfully isolated by SELEX.This new method can be used for the identification of BMSCs.In addition,this idea hopefully will be applied to create an aptamer-coated scaffold for application in tissue engineering to recruit circulating stem cells.