应用抑制性消减杂交技术筛选三氧化二砷对肝细胞调节的差异表达基因  

Screening and identification of target genes differentially expressed in HepG2 cells treated with arsenic trioxide by suppression subtractive hybridization technique

作　　者：吴顺华[1] 成军[1] 郑玉建[2] 张跃新[2] 刘妍[1] 郭江[1] 张黎颍 王国荃[2] 

机构地区：[1]北京地坛医院传染病研究所,北京市100011 [2]新疆医科大学公共卫生学院环境与基因研究室

出　　处：《世界华人消化杂志》2005年第13期1535-1539,共5页World Chinese Journal of Digestology

基　　金：国家自然科学基金攻关项目;No.C03011402;No.C30070689军队"九五"科技攻关项目;No.98D063军队回国留学人员启动基金项目;No.98H038军队"十五"科技攻关青年基金项目;No.01Q138军队"十五"科技攻关面上项目;No.01MB135~~

摘　　要：目的:应用抑制性消减杂交(suppression subtractive hybridization,SSH)技术构建As2O3处理的人肝癌细胞系HepG2差异表达基因的cDNA消减文库,筛选并克隆As2O3调节相关基因,阐明As2O3对肝细胞调节作用的分子生物学机制. 方法:以As2O3处理HepG2细胞,同时以PBS处理的相同细胞系作为对照;24 h后制备细胞裂解液,提取mRNA并逆转录为cDNA,经RsaI酶切后,将实验组cDNA分成两组,分别与两种不同的接头衔接,再与对照组cDNA进行两次消减杂交及两次抑制性多聚酶链反应(PCR),将产物与T/A载体连接,构建cDNA 消减文库,并转染大肠杆菌J109进行文库扩增,随机挑选克隆PCR扩增后进行测序及同源性分析. 结果:成功构建三As2O3处理HepG2细胞差异表达基因的cDNA消减文库.文库扩增后得到109个白色克隆,进行菌落PCR分析,均得到200-1000bp插入片段.挑取含有插入片段的36个克隆进行测序,并通过生物信息学分析获得15种已知基因序列和6个未知功能的新基因. 结论:应用SSH技术成功构建了As2O3处理HepG2细胞差异表达基因的cDNA消减文库.AIM: To clone and identify human genes differentially expressed in human hepatocarcinoma HepG2 cells treated with arsenic trioxide by constructing a subtractive cDNA library with suppression subtractive hybridization (SSH) technique, and to elucidate the molecular mechanism of arsenic trioxide in the regulation of liver cells. METHODS: The mRNA was isolated from HepG2 cells treated with arsenic trioxide and PBS, respectively, and then cDNA was synthesized. After digestion of restriction enzyme Rsa\, small sizes cDNA were obtained. Then the tester cDNA was subdivided into two portions and each was ligated with a different cDNA adaptor. After the tester cDNA was hybridized with driver cDNA (twice) and underwent nested polymerase chain reaction (PCR) (twice), the DNA fragment was subcloned into T/A plasmid vectors to establish the subtractive cDNA library. Amplification of the library was performed in E. coli strain JM109. The amplified cDNA was sequenced and analyzed in GenBank with BLAST search after colony PCR. RESULTS: The subtractive cDNA library of genes differentially expressed in HepG2 cells treated with arsenic trioxide was constructed successfully. The amplified library contained 109 positive clones. Colony PCR showed that these clones all contained 200-1 000 bp inserts. Thirty-six clones were analyzed by sequencing and bioinformatics. The results showed there were 15 coding sequences with known function and 6 novel ones with unknown function. CONCLUSION: A subtractive cDNA library of genes differentially expressed in HepG2 cells treated with arsenic trioxide was constructed successfully using SSH technique.

关 键 词：差异表达基因 抑制性消减杂交技术 三氧化二砷 CDNA消减文库 细胞调节 人肝癌细胞系HEPG2 筛选 HepG2细胞 As2O3 分子生物学机制 生物信息学分析 多聚酶链反应 文库扩增 插入片段 细胞裂解液 同源性分析 PCR扩增 PCR分析 

分 类 号：R735.7[医药卫生—肿瘤] R373.21[医药卫生—临床医学]