SELEX技术筛选乳腺癌亚型组织细胞的DNA适配子  

Screening of DNA aptamers for subtype of breast cancer cells by using SELEX

作　　者：潘先均 刘美 李光新 徐发良 PAN Xianjun;LIU Mei;LI Guangxin;XU Faliang(Breast Cancer Center,Chongqing 400030,China;Department of Pathology,Chongqing University Cancer Hospital)

机构地区：[1]重庆大学附属肿瘤医院乳腺肿瘤中心,重庆400030 [2]重庆大学附属肿瘤医院病理科,重庆400030

出　　处：《南京医科大学学报(自然科学版)》2025年第2期147-156,共10页Journal of Nanjing Medical University(Natural Sciences)

基　　金：重庆市自然科学基金(cstc2020jcyj-zdxmX0030);重庆市社会事业与民生保障专项课题(cstc2015shmszx120051)。

摘　　要：目的:获取结合于不同亚型乳腺癌组织细胞的DNA适配子。方法:采用PCR法建立随机双链DNA(double-stranded DNA,dsDNA)文库,用卵白素包被的琼脂糖珠从dsDNA库获取单链DNA(single-stranded DNA,ssDNA)文库;以消减细胞-指数富集的配体进化系统(systematic evolution of ligands by exponential enrichment,SELEX)技术为基础,将体外培养的乳腺癌细胞MCF-7和MDA-MB-231交替作为靶标进行正筛选,以相同组织来源的乳腺上皮细胞MCF-10A进行负筛选,琼脂糖电泳法检测PCR扩增和适配子回收、温度梯度实验优化PCR条件、流式细胞术监测适配子与靶细胞的亲和力,采用常规分子生物学技术完成适配子的克隆和测序,通过序列分析初步筛选适配子;综合采用流式细胞术、激光共聚焦和免疫荧光染色等实验优选DNA适配子。结果:成功建立ssDNA文库,优化了SELEX筛选条件,通过20轮交叉串联的细胞-SELEX筛选获取了特异性结合乳腺癌MCF-7和MDA-MB-231细胞的DNA适配子,随机挑选100个阳性克隆测序,鉴定出72个DNA适配子,采用Blastn程序进行精确比对未发现相似序列。通过序列分析初步筛选出适配子5个,经优选实验获取了可结合乳腺癌亚型组织细胞的适配子2个。结论:采用交叉串联的细胞-SELEX技术,可在较短时间获取结合不同临床乳腺癌亚型组织细胞的DNA适配子,为乳腺癌靶向递药系统的探索研究提供了可能。Objective:To get DNA aptamers binding to different subtypes of breast cancer cells.Methods:A random double⁃stranded DNA(dsDNA)pool was established by PCR,and single⁃stranded DNA(ssDNA)library was separated from the dsDNA pool by coated sepharose.Based on subtractive cell⁃systematic evolution of ligands by exponential enrichment(SELEX),the in vitro cultured breast cancer cells,MCF⁃7 and MDA⁃MB⁃231,were alternately used as positive screening targets while normal mammary gland cell MCF⁃10A as negative.The effect of PCR amplification and the recovery of aptamer were confirmed by agarose electrophoresis.The PCR conditions were optimized through temperature gradient experiment,while the affinity of aptamers for target cells was monitored by using flow cytometry(FCM).Cloning and sequencing of aptamers were carried out by conventional molecular biology techniques.The aptamers were preliminarily screened by sequence analysis,and DNA aptamers were selected by FCM,laser confocal and immunofluorescence staining.Results:A random ssDNA library was successfully established,and the screening conditions of SELEX were optimized.Specific DNA aptamers for MCF⁃7 and MDA⁃MB⁃231 cells were obtained after 20 runs’tandem crossed cell⁃SELEX.Of 100 positive sequenced clones,72 aptamers were identified.Accurate alignment of Blastn implied no similar sequence to the 72 submitted aptamers.Five aptamers were preliminarily screened by sequence analysis,and two aptamers which could bind to breast cancer subtype tissue cells were obtained by optimization experiment.Conclusion:By using the tandem crossed cell⁃SELEX,DNA aptamers of broad⁃spectrum combing with clinical breast cancer subtypes can be obtained in a short time,which may be helpful for the targeted drug delivery system of breast cancer.

关 键 词：乳腺癌 SELEX 适配子 交叉串联 广谱 

分 类 号：R737.9[医药卫生—肿瘤]