摘要
目的利用细胞指数富集配基的系统进化技术(Cell-SELEX)筛选去势抵抗性前列腺癌(CRPC)细胞特异性核酸适配体。方法以人CRPC细胞C4-2基因组作为正筛选细胞,以激素依赖性前列腺癌细胞LNCap基因组作为负筛选细胞进行Cell-SELEX筛选。PCR上游引物的5’端标记FITC,下游引物的5’端标记生物素,将每一轮次筛选得到的DNA产物为模板进行PCR扩增、纯化。用生物素-链霉亲和素磁珠分离法将PCR产物分离后获得的产物用于下一轮次的筛选,用流式细胞仪监测Cell-SELEX筛选进程;将最后一轮次筛选所得的DNA产物行PCR扩增纯化、克隆及测序,预测核酸适配体的二级结构,计算适配体的平衡解离常数。用流式细胞术、细胞免疫荧光及组织免疫荧光检测适配体对CRPC组织细胞的特异性结合。结果随着Cell-SELEX的筛选进程,DNA产物与CRPC细胞的结合率逐渐增加,至第14轮达到最高。DNA结构预测分析显示,核酸适配体CRPC-1和CRPC-2的二级结构主要为茎环结构。流式细胞术和细胞免疫荧光实验显示,CRPC-1和CRPC-2可特异性靶向CRPC细胞C4-2;组织免疫荧光实验显示,CRPC-1能特异性识别并结合CRPC组织。结论Cell-SELEX可筛选CRPC细胞特异性核酸适配体,对靶向诊断技术的研发和及时调整临床治疗方案、改善患者预后具有重要意义。
Objective To screen nucleic acid aptamers that can specifically recognize castration-resistant prostate cancer(CRPC)cells by Cell-SELEX.Methods For Cell-SELEX selection,CRPC cell lines C4-2 were used as positive control cells,while androgen-dependent prostate cancer cell lines LNCap were used as negative control cells.The 5’end of the upstream primers was labeled with FITC,and the 5’end of the downstream primers was labeled with biotin.Single-stranded DNA(ssDNA)collected from each round of screening was used as template for PCR amplification and purification.The biotin-streptavidin magnetic bead separation was used to isolate PCR product for the next round of screening.The process of Cell-SELEX was analyzed by flow cytometry.ssDNA products of the last round were collected for PCR amplification,purification,cloning and DNA sequencing.The secondary structure of selected DNA aptamers was predicted.Dissociation constants of the two aptamers were calculated.Flow cytometry and confocal microscopy were used to evaluate selective binding of aptamers to CRPC cells and tissues.Results The binding rate of DNA products to CRPC cells gradually increased with the increase of selection cycles,reaching the highest in the last round.DNA structure prediction analysis showed that the secondary structure of aptamers CRPC-1 and CRPC-2 was mainly stem-loop structure.Flow cytometry analysis and confocal images showed that both CRPC-1 and CRPC-2 could specifically target C4-2 cells.In addition,immunohistofluorescence assay showed that CRPC-1 could specifically target CRPC tissues.Conclusion Cell-SELEX can be used to screen aptamers that specifically target CRPC cells and tissues,which provides experimental basis for early screening and targeted diagnosis of CRPC.It is of significance for treatment plan adjustment and prognosis improvement of prostate cancer.
作者
仲津漫
丁健科
邓蕾
向颖
刘朵朵
杨全新
ZHONG Jinman;DING Jianke;DENG Lei;XIANG Ying;LIU Duoduo;YANG Quanxin(Department of Radiology,The Second Affiliated Hospital of Xi’an Jiaotong University,Xi’an 710004;Deportment of Plastic Surgery,Xijing Hospital of Air Force Medical University,Xi’an 710032,China)
出处
《西安交通大学学报（医学版）》
CAS
CSCD
北大核心
2021年第5期687-692,共6页
Journal of Xi’an Jiaotong University（Medical Sciences）
基金
陕西省重点研发计划一般项目(No.2021SF-267)。
