骨肉瘤细胞适配体高质量制备体系的建立  

Establishment of a high quality preparation system for osteosarcoma cell aptamers

作　　者：魏唐文 梁骥 张宇飞 王华[4] 王秀娟 魏兵 WEI Tangwen;LIANG Ji;ZHANG Yufei;WANG Hua;WANG Xiujuan;WEI Bing(College of Public Health,Guilin Medical University,Guilin 541199,China;College of Laboratory Medicine,Guilin Medical University,Guilin 541199,China;Department of Academic Affairs,Guilin Medical University,Guilin 541199,China;Affiliated Hospital of Beihua University,Jilin 132011,China;Affiliated Hospital of Guilin Medical University,Guilin 541001,China)

机构地区：[1]桂林医学院公共卫生学院,桂林541199 [2]桂林医学院医学检验学院,桂林541199 [3]桂林医学院教务处,桂林541199 [4]北华大学附属医院,吉林132011 [5]桂林医学院附属医院,桂林541001

出　　处：《华夏医学》2025年第2期48-54,共7页Acta Medicinae Sinica

基　　金：广西肿瘤免疫与微环境调控重点实验室2024年度自主课题(2024ZZ003);广西医疗卫生重点培育学科建设项目(桂卫科教发〔2023〕1号)。

摘　　要：目的探索肿瘤细胞适配体高质量制备体系建立的可行性。方法构建适配体制备体系:基于细胞-SELEX(Cell-SELEX)经典方法,选用骨肉瘤U2-OS细胞株作为正向筛选靶细胞,人纤维肉瘤HT-1080细胞株作为反向筛选细胞,制备U2-OS适配体文库。从荧光光谱和流式细胞术两种方法中筛选适配体文库制备进程的最佳监测方法。适配体文库扩增体系条件优化。对最终制备的适配体文库与靶细胞结合的特异性进行研究。结果监测方法筛选结果显示,荧光光谱技术更适合作为适配体文库制备的监测技术。适配体文库扩增体系优化结果显示,在12.5μL PCR的扩增体系中,模板量40~41 ng、循环数12~14可作为优化扩增条件。新体系制备的终端文库与两种细胞株特异性鉴定结果显示,目标适配体对U2-OS细胞具有高度亲和性与特异性。结论正向筛选伴随反向筛选、荧光光谱技术以及PCR模板浓度定量优化成功构建骨肉瘤细胞适配体高质量制备体系,为其他肿瘤适配体高质量制备提供了新的思路与途径。Objective To explore the feasibility of establishing a high-quality preparation system for tumor cell aptamers.Methods The aptamer preparation system was constructed based on the classic cell-SELEX(Cell-SELEX)method.The osteosarcoma U2-OS cell line was selected as the forward screening target cell,while the human fibrosarcoma HT-1080 cell line served as the reverse screening cell.The U2-OS aptamer library was prepared,and the optimal monitoring method for aptamer library preparation progress was evaluated using fluorescence spectroscopy and flow cytometry.Additionally,the conditions for optimizing the aptamer library amplification system were established,and the specificity of the final aptamer library binding to target cells was investigated.Results The results from the monitoring method screening indicated that fluorescence spectroscopy is more suitable for monitoring aptamer library preparation.Optimization of the aptamer library amplification system revealed that a 12.5μL PCR amplification system with a template amount of 40 to 41 ng and a cycle number of 12 to 14 represents the optimized amplification conditions.The terminal library prepared by the new system,along with specific identification results from the two cell lines,demonstrated that the target aptamer exhibits high affinity and specificity for U2-OS cells.Conclusion The combination of forward screening with reverse screening,the use of fluorescence spectroscopy technology,and the quantitative optimization of PCR template concentration successfully established a high-quality preparation system for osteosarcoma cell aptamers.This approach provides new insights and methodologies for the high-quality preparation of other tumor aptamers.

关 键 词：适配体 肿瘤细胞 流式细胞技术 荧光光谱 

分 类 号：R738.1[医药卫生—肿瘤]