上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 技术与方法 • 上一篇    

利用细胞－指数式富集的配体系统进化技术筛选肿瘤细胞DNA适配子

徐发良1，周 琦1，匡 毅1，陈小乐1，张海伟1，顾长国2   

  1. 1.重庆市肿瘤研究所 重庆市肿瘤医院乳腺中心， 重庆 400030； 2.解放军第452医院检验科， 成都 610061
  • 出版日期:2013-12-28 发布日期:2014-01-02
  • 作者简介:徐发良（1970—）， 男， 副教授， 副主任医师， 硕士生导师; 电子信箱: flxu88@163.com。
  • 基金资助:

    重庆市科学技术委员会科技攻关项目(CSTC, 2010AC5132)；重庆市卫生局医学科研项目(2009-2-122)

Screening of DNA aptamers for recognizing tumor cells by using cell-systematic evolution of ligands by exponential enrichment

XU Fa-liang1, ZHOU Qi1, KUANG Yi1, CHEN Xiao-le1, ZHANG Hai-wei1, GU Chang-guo2   

  1. 1.The Center of Breast Disease, Chongqing Cancer Institute and Chongqing Cancer Hospital, Chongqing 400030, China; 2.Clinical Laboratory, 452 Hospital of
    PLA, Chengdu 610061, China
  • Online:2013-12-28 Published:2014-01-02
  • Supported by:
    Key Program of Science and Technology Commission of Chongqing Municipality, 2010AC5132; Medical Research Project of Chongqing Municipal Health Bureau, 2009-2-122

PDF (PC)

2692

摘要：

目的 建立一种通用的细胞-指数式富集的配体系统进化（Cell-SELEX）技术平台，用于筛选特异性结合于肿瘤细胞的DNA适配子。方法 采用PCR法建立随机双链DNA（dsDNA）寡核苷酸文库，用卵白素包被的琼脂糖珠从dsDNA库分离单链DNA（ssDNA）文库；以体外培养的肿瘤细胞作为正筛选的靶标, 以相同组织来源的正常对照细胞作为负筛选的靶标，消减法进行Cell-SELEX筛选和PCR扩增以获取DNA适配子；流式细胞仪监测适配子的富集效率（亲和力）；采用常规分子生物学技术进行适配子的克隆、测序和序列分析。结果 成功建立了ssDNA库，经过12轮Cell-SELEX筛选，获得了特异性结合于肿瘤细胞的DNA适配子；挑选120个阳性克隆送测序，鉴定出21个适配子，采用NCBI网站提供的Blastn程序进行精确比对未发现相似序列。结论 本实验建立的技术平台可在较短时间内获取特异性结合于特定肿瘤细胞的DNA适配子。

关键词: 细胞-指数式富集的配体系统进化, 适配子, 肿瘤, 靶向诊断, 靶向治疗

Abstract:

Objective To establish commonly used technical platform of cell-systematic evolution of ligands by exponential enrichment (cell-SELEX) for screening DNA aptamers specifically binding to tumor cells. Methods A random double-stranded DNA (dsDNA) pool was established by PCR, and single-stranded DNA (ssDNA) library was then separated from dsDNA pool by using avidin coated Sepharose. In order to get DNA aptamers for cancer cells, cell-SELEX and PCR were performed by using cultured gastric cancer cell line as positive screening target while normal gastric mucosal cell line as negative. Flow cytometry (FCM) was used to monitor the enrichment efficiency and affinity of aptamers. Cloning, sequencing, and sequence analysis of aptamers by conventional molecular biology techniques were also carried out. Results A random ssDNA pool was successfully established and specific aptamers of tumor cells were got after 12 runs' cell-SELEX. Of 120 positive sequenced clones, 21 aptamers were identified. Accurate alignment of Blastn implied that not any similar sequence to 21 submitted aptamers was found in NCBI data base. Conclusion The article provided a technical platform for obtaining DNA aptamers specifically binding to tumor cells in a relatively short period of time.

Key words: cell-systematic evolution of ligands by exponential enrichment, aptamer, tumor, targeting diagnosis, targeted therapy