骨肉瘤特异生物诊断探针制备  

Preparation of biological probe special for osteosarcoma

作　　者：王华[1] 梁骥 王法佳 周磊 马艳[1] 张宇飞 傅桂莲[4] WANG Hua;LIANG Ji;WANG Fa-Jia(Affiliated Hospital of Beihua University,Jilin 132000,Jilin,China)

机构地区：[1]北华大学附属医院,吉林吉林132000 [2]广西桂林医学院 [3]北华大学研究生院 [4]北华大学检验学院

出　　处：《中国老年学杂志》2020年第2期399-403,共5页Chinese Journal of Gerontology

基　　金：吉林省教育厅资助项目([2016]No63);广西自然科学基金(2018GXNSFAA138035)

摘　　要：目的构建与骨肉瘤细胞高亲和性与特异性结合的DNA寡核苷酸适配体库,用于骨肉瘤临床特异性诊断。方法体外合成40 nt高通量随机序列的单链DNA寡核苷酸文库(ssDNAs);骨肉瘤细胞(U-2 OS)作为目标细胞与文库ssDNAs反应;指数富集配体的系统进化(SELEX)技术用于筛选富集与靶细胞结合的寡核苷酸;聚合酶链反应(PCR)技术与亲和层析技术用于次轮反应文库的制备;应用荧光光谱跟踪筛选过程;对最后的筛选序列进行克隆、测序与保存。结果十一轮至十三轮筛选后的ssDNAs荧光光谱曲线显示了有意义重叠,因此结束筛选;对十三轮筛选的ssDNAs进行了克隆测序,依据一级结构的同源序列建立了寡核苷酸探针组群。结论应用SELEX技术最终从高通量的随机文库中获得了与U-2 OS具有高度亲和性的寡核苷酸适配体,适配体库的建立不仅仅用于骨肉瘤的特异性诊断,也将为探针-载体-抗肿瘤药物复合体的研制与实施骨肉瘤定向治疗搭建了可行性平台。Objective To establish a oligonucleotides DNA aptamer library that could bind to osteosarcoma cells with high affinity and specificity for clinical diagnosis.Methods High-throughout oligonucleotides DNA library containing 40 nt random sequences were synthesized in vivo.The osteosarcoma(U-2 OS)cells as for positive target cells were incubated with oligonucleotides DNA library(ssDNAs).Screening and exponential enrichment of ssDNA combined with target cells were finished through the SELEX technology.The secondary libraries were prepared by PCR and affinity chromatography technique.Screening process was monitored with fluorescence spectrum.The oligonucleotides from the thirteenth round selection were cloned,sequenced and collected.Results Screening was ended when fluorescence spectrum curves of the eleventh and thirteenth round selection showed significant overlapping.The ssDNAs from the thirteenth round selection were cloned and sequenced.Aptamer library had been set up according to the primary homologous structure.Conclusions Oligonucleotides library specific binding to U-2 OS cells has been obtained through thirteen rounds selection.Aptamers of U-2 OS cells are not only used for diagnosis,it’s also possible to establish a platform of target therapy by using complex of aptamer-vector-drug to avoid damage of chemotherapy.

关 键 词：指数富集配体的系统进化(SELEX) 骨肉瘤细胞(U-2 OS) 探针 克隆 测序 

分 类 号：R604[医药卫生—外科学]