文章摘要
郑 岳,邸 娅,赵运旺,卢坤玲,黄百海,徐 欢.肝癌血清肿瘤标志物适配体的筛选及其亲和力和特异性的鉴定[J].南京医科大学学报,2018,(10):1350~1356
肝癌血清肿瘤标志物适配体的筛选及其亲和力和特异性的鉴定
Screening of liver cancer serum tumor markers’ aptamers and identification of their affinity and specificity
投稿时间:2018-03-04  
DOI:10.7655/NYDXBNS20181004
中文关键词: 原发性肝癌  血清  SELEX  适配体
英文关键词: primary liver cancer  serum  SELEX  aptamer
基金项目:
作者单位
郑 岳 秦皇岛市第一医院消化内科河北 秦皇岛 066000 
邸 娅 秦皇岛市第一医院消化内科河北 秦皇岛 066000 
赵运旺 秦皇岛市第一医院消化内科河北 秦皇岛 066000 
卢坤玲 秦皇岛市第一医院消化内科河北 秦皇岛 066000 
黄百海 燕山大学环境与化学工程学院河北 秦皇岛 066004 
徐 欢 秦皇岛市第一医院消化内科河北 秦皇岛 066000 
摘要点击次数: 210
全文下载次数: 261
中文摘要:
      目的:采用指数富集的配基系统进化技术(SELEX)筛选获得一组已知和未知肝癌血清肿瘤标志物的适配体,为肝癌的早期诊断提供新的便捷方法。方法:收集50例首次经诊断证实为原发性肝癌的患者血清,以及50例体检无异常的正常人血清,并分别等比例混合制备成肝癌混合血清和正常人混合血清,作为筛选的靶标分子,磁珠作为分离载体。先将磁珠?正常人血清复合物与ssDNA文库结合,取上清再与磁珠?肝癌血清结合,经过洗脱、分离与肝癌血清结合的特异性ssDNA,并进行扩增,链霉亲和素磁珠法制备次级ssDNA,进行9轮筛选,将9轮筛选获得的饱和文库与PMD18?T载体连接进行转化、挑选单克隆送上海生工进行测序,同时利用流式细胞术测定肝癌血清肿瘤标志物适配体的亲和力(Kd值)。结果:经9轮筛选,成功分离出200个核酸序列,其中序列不同的有10个。特异性检测表明,筛选得到的肝癌血清肿瘤标志物适配体与肝癌血清的结合解离常数均在纳摩尔级水平,其中Seq?1、Seq?16、Seq?17、Seq?56、Seq?72号适配体能高特异性结合肝癌血清,与正常人血清不结合,再通过200例肝癌血清和200例正常人血清进一步鉴定5条肝癌血清肿瘤标志物适配体检出肝癌的阳性率,阳性检出率达91%以上。结论:利用随机单链寡核苷酸文库成功获得与肝癌患者血清特异性结合的适配体,所获得的适配体具有拮抗肝癌血清的能力,有可能为肝癌的早期诊断提供新的便捷方法。
英文摘要:
      Objective:To obtain a set of aptamers of liver cancer serum markers by SELEX screening,and to provide new molecular biological detection method for the early diagnosis of liver cancer. Methods:Serum samples from 50 patients with primary hepatocellular carcinoma(HCC) and 50 normal serum samples of which physical examination showed no abnormalities were collected and mixed in equal proportions to prepare mixed sera of HCC and normal subjects. The serum was used as the target molecule for screening. Magnetic beads?normal human serum complex was combined with ssDNA library. The supernatant was then combined with magnetic beads?liver cancer serum to elute and isolate the specific ssDNA binding to serum of liver cancer. Nine rounds of ssDNA were screened by the method of the streptavidin biotin method,and transformed into PMD18?T vector. The single clones were selected and sequenced by Shanghai Biosystems. Affinity determination of serum tumor marker aptamers in patients was performed by flow cytometry. Results:After 9 rounds of screening,200 nucleic acid sequences were successfully isolated,of which 10 sequences were different. Specificity test showed that the binding dissociation constants of serum tumor marker aptamers and HCC serum were all in nanomolar level. Among them,Seq?1,Seq?16,Seq?17,Seq?56,Seq?72 aptamers could be bound to the serum of liver cancer with highly specificity,and could not bound to the normal serum. The positive rate of serum tumor marker aptamers was detected in 200 cases of liver cancer serum and 200 cases of normal human serum,positive detection rate was more than 91%. Conclusion:The use of a random single?stranded oligonucleotide library to successfully obtain an aptamer that specifically binds to liver cancer patient serum,and the obtained aptamer has the ability to antagonize liver cancer serum,which may provide a new convenient method for the early diagnosis of liver cancer.
查看全文   查看/发表评论  下载PDF阅读器
关闭