用SSH方法构建BTCC患者尿脱落细胞差异表达基因的cDNA消减文库  

Constructing the SSH cDNA library in urine exfoliated urothelial cells of bladder neoplasms and normal

作　　者：郭柏鸿[1] 车团结[2] 张冲[1] 李琳[2] 史葆光[1] 王建伟[1] 陈一戎[1] 

机构地区：[1]甘肃省人民医院泌尿外科,兰州730000 [2]兰州大学生命科学学院细胞生物学研究所

出　　处：《临床泌尿外科杂志》2008年第3期222-227,共6页Journal of Clinical Urology

摘　　要：目的:应用抑制性消减杂交(SSH)方法构建膀胱移行细胞癌(BTCC)患者与正常人尿脱落细胞差异表达基因cDNA消减文库。方法:分别从BTCC患者与正常人尿液中提取总mRNA,用SMART技术反转录成cDNA,经过HaeⅢ酶切后将BTCC尿脱落细胞cDNA分为两组并接上接头,再与过量正常膀胱尿脱落细胞cD-NA进行两轮消减杂交及两轮抑制性聚合酶链反应(PCR),使得差异表达的DNA片段得以富集。PCR产物与T/A载体连接并转化大肠杆菌JM109构建成差异表达基因的cDNA消减文库。文库扩增后,随机挑取克隆进行酶切、测序及同源性分析。结果:PCR鉴定有317个克隆载有主要在200～900bp之间呈随机分布的插入片段,片段插入率达82.6%,证实建库成功。对20个质粒测序结果经同源性比对分析,其中20个片段源于17个已知基因,1个克隆在GenBank中未检索到与其有相似性的基因序列,表明它们可能为BTCC差异表达的新基因。结论:该消减杂交文库质量可靠,其成功构建为进一步筛选、克隆BTCC差异表达基因提供了依据。Objective： To construct the SSH cDNA library from exfoliated urothelial cells of bladder neoplasms and normal. Methods： Total mRNA was extracted from BTCC and normal exfoliated urothelial cells in urine respectively. Then double-strand cDNA were synthesized and restricted by Haem. BTCC cDNA were divided into two groups and ligated with either adaptor 1 or adaptor. After hybridized with normal exfoliated urothelial cells cDNA twice and underwent nested PCR, the PCR products were cloned into PGM-T vector and transformed to E. coli JM109. Some positive clones randomly picked up were digested, sequenced and homologously analyzed. Resuits：The SSH library contained about 400 positive clones. Random analysis of 384 clones with enzyme restriction showed that 317clones contained cDNA fragments which were mainly between 200-900 bp. The inserted rate reached to 82.6%. A subtracted cDNA library of differentially expressed genes is successfully constructed with SSH. Among 20 arbitrary clones derived from the above 317 clones, No. 243 clone is a previously unknown sequence and the other 17clones were derived from 19 known genes. Conclusions： The quality of the SSH library of BTCC is reliable and its construction would lay the foundation for further screening differentially expressed genes.

关 键 词：膀胱移行细胞癌 基因差异表达 cDNA文库 抑制性消减杂交 

分 类 号：R737.14[医药卫生—肿瘤]