摘要
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(apt),探讨apt与BMSCs的结合方式及能力。方法利用SELEX技术,以大鼠BMSCs为靶点,从构建的核酸文库中反复筛选、克隆、测序及分析,获得候选apt。流式细胞术测定和软件分析获得不同浓度(0、4、8、16、32、64、128、256 nmol/L)候选apt与BMSCs的结合曲线及平衡解离常数。胰酶消化实验探究apt与BMSCs结合的靶点,并观察apt体外捕获BMSCs的能力。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。经序列分析适配子apt7与BMSCs的亲和力最高,平衡解离常数为(11.79±0.72)nmol/L。胰酶消化实验表明当胰酶消化细胞时间超过10 min后,apt7与BMSCs的亲和力下降甚至消失。体外捕获实验表明固定于培养皿上面的apt7能体外捕获骨髓细胞液中的大鼠BMSCs。结论本研究通过SELEX技术成功分离出能高亲和力、高特异性结合大鼠BMSCs的特异性适配子apt7,为进一步将适配子应用于组织工程支架表面招募循环干细胞研究提供了实验依据。
Objective To generate specific aptamers from bone marrow mesenchymal stem cells(BMSCs)of rats.And the binding mode and capture ability of the aptamer with the BMSCs in vitro were investigated.Methods Systematic evolution of ligands by exponential enrichment(SELEX)technology was applied to obtain aptamers binding to BMSCs of rats.After the enrichment of the nucleic acid sequence specific binding to BMSCs of rat DNA pool was cloned and sequence analysis was helped to obtain candidate aptamers.Flow cytometry and confocal imaging showed the binding affinity between aptamers and BMSCs.Candidate aptamers were obtained from cloning,sequencing and sequence analysis on the most enriched library.Different concentrations of candidate aptamers were incubated with BMSCs,then mean fluorescence intensity was calculated and applied to the formula to generate Kd by SigmaPlot software.Trypsin digestion test was applied to explore whether the aptamers could bind to the membrane protein on BMSCs.And we added the biotinylated aptamers to a petri dish which was coated with streptavidin and incubated overnight.The capture capability of aptamers to BMSCs was observed.Results Flow cytometry and confocal imaging showed that apt7 had the best affinity to BMSCs with a Kd of only(11.79±0.72)nmol/L.After the cells were trypsinized for more than 10 min,the affinity between apt7 and BMSCs decreased or disappeared.The capturing assay showed that apt7 could capture BMSCs in vitro.Conclusion In this study,SELEX technology successfully isolated specific apt7 from BMSCs of rats.Apt7 showed high affinity and specificity to BMSCs and could capture BMSCs in vitro.Furthermore,Apt7 counld be applied to create an aptamer-coated scaffold to recruit circulating stem cells in tissue engineering.
作者
洪昊
李昭
董念国
Hong Hao;Li Zhao;Dong Nianguo(Department of Cardiovarscular Surgery,Union Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430022,China;Department of Cardiovarscular Surgery,Fuwai Central China Cardiovascular Hospital,Zhengzhou 45146,China)
出处
《中华实验外科杂志》
CAS
北大核心
2021年第8期1470-1474,共5页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金面上项目(81873502)
华中科技大学自主创新研究基金项目(2017KFYXJJ250)。
