人骨肉瘤细胞适配体的早期筛选  

Early Screening of Human Osteosarcoma Cell Aptamer

作　　者：王华[1] 梁骥[2] 高远[3] 孙娜[3] 马艳[1] 高丽君[3] 夏薇[3] 

机构地区：[1]北华大学附属医院,吉林吉林132011 [2]吉林大学第一临床学院,吉林长春130021 [3]北华大学医学检验学院,吉林吉林132013

出　　处：《北华大学学报（自然科学版）》2015年第3期311-315,共5页Journal of Beihua University（Natural Science）

基　　金：吉林省教育厅科学技术研究项目(2011132)

摘　　要：目的体外随机合成单链寡核苷酸备选文库,以骨肉瘤U-2 OS完整细胞作为靶点,筛选出与其结合的高特异性、高亲和性单链DNA(ss DNA).方法将体外合成随机碱基序列的单链DNA文库与人骨肉瘤U-2 OS细胞相结合,收获与靶细胞结合的单链DNA,通过优化PCR条件进行大量扩增后,应用生物素-链霉亲和素亲和层析柱分离出正链DNA.结果以10,14以及18个循环时扩增ds DNA产物最理想;应用生物素-链霉亲和素磁珠分离法制备的正链ss DNA纯度高,可有效保证次级文库的筛选.结论应用Cell-SELEX技术可成功筛选早期ss DNA文库,为后续获得针对人骨肉瘤U-2 OS细胞的高特异性适配体奠定实验基础.Objective Prepare single-stranded oligonucleotide （ssDNA） with high affinity and specificity for Osteosarcoma U-2OS, a target cell, by exponential enrichment strategy. Method The designed single-stranded DNA library were synthesized in vitro. After incubation of DNA library pool with U-2OS ceils, the ssDNAs binding to the target cells U-2OS were amplified by PCR that optimum cycle had been determined. After amplification, separation ssDNA from dsDNA was finished by affinity chromatography with streptavidin-coated Sepharose beads. Results The products of dsDNA from 10,14 and 18 cycle showed desirable image. The single- stranded DNA （ssDNA） separated from dsDNA by Streptavidin-coated Sepharose beads was of high purity, which was next round selection. Conclusion We have successfully selected the initial ssDNA pool by Cell-SELEX, which will be used for the development of DNA aptamer with high affinity for U-2OS Osteosarcoma cell.

关 键 词：人骨肉瘤 Cell-SELEX技术 适配体 

分 类 号：R738.1[医药卫生—肿瘤]