应用抑制性消减杂交技术分离E1A药敏相关基因  

Isolation of E1A-Related Drug-sensitive New Genes by Suppression Subtractive Hybridization

作　　者：马业伟[1] 千新来[1] 赵清正[1] 周小山[1] 李艳春[1] 

机构地区：[1]中国协和医科大学中国医学科学院肿瘤医院肿瘤研究所,北京100021

出　　处：《癌症》2004年第2期146-149,共4页Chinese Journal of Cancer

基　　金：国家自然科学基金项目(No.30171058)~~

摘　　要：背景与目的:大量研究已经证明,腺病毒5型E1A基因能抑制肿瘤的生长和转移,逆转其恶性表型,特别是能提高肿瘤细胞对多种化疗药物的敏感性,对射线也具有明显的增敏作用。但E1A基因通过哪些基因发挥作用还不知道。本研究利用抑制性消减杂交(suppressionsubtractivehybridization,SSH)技术在药物敏感的肿瘤细胞中分离差异表达的E1A药敏相关基因。方法:以E1A蛋白处理的人头颈淋巴结转移肿瘤细胞LN686为实验组,以亲本LN686细胞为对照组,提取mRNA,构建消减cDNA文库。随机挑取克隆,采用斑点杂交技术鉴定差异基因片段的表达水平,分析所获cDNA核苷酸序列,并在GenBank中进行比较,半定量RT-PCR鉴定实验组和对照组中新基因的mRNA水平。结果:文库包含约7000个阳性克隆,随机挑取384个克隆进行PCR鉴定,其中362个有插入片段,通过斑点杂交技术,证实这些基因在实验组中表达水平较对照组显著提高。将这362个克隆进行测序并经过BLAST分析,表明10个为新基因,已在GenBank中登录。对其中7个新基因进行半定量RT-PCR检测,证明经E1A蛋白处理的LN686细胞中,新基因的表达水平明显高于亲本LN686细胞,相差3～8倍。结论:应用抑制性消减杂交技术筛选到10个新基因片段,为进一步克隆其全长基因、研究其功能打下基础。BACKGROUND &OBJECTIVE: It has been well demonstrated that E1A, as a tumor suppression gene, is capable of inhibiting the growth and metastasis of different tumors, and reversing the malignant phenotype. Particularly, the gene possesses the ability to greatly enhance the drug sensitivity of tumor cells to several antitumor agents, and also increase the radio sensitivity. However, the associated genes through which E1A can exert its antitumor functions still remain unknown. The aim of this study was to isolate E1A anticancer related genes,which were differentially expressed in drug sensitive tumor cells using suppression subtractive hybridization (SSH). METHODS: To construct SSH library of human lymph node metastasis tumor cells (LN686) using the mRNA from LN686 cells treated by E1A protein and the parental LN686 cells as tester and driver, respectively. Positive clones in the library were selected randomly, and dot blot was used for the analysis of expression pattern of the differentially expressed gene fragments. The sequences of cDNA fragments were analyzed and compared with that in GenBank. The mRNA levels of the novel genes in tester and driver were determined by semi quantitative RT PCR analysis. RESULTS: The SSH library contained about 7000 positive clones. Random analysis of 384 clones with PCR demonstrated that 362 clones contained inserted fragments. The consequence of dot blot demonstrated that these genes were over expressed in the tester compared to the driver significantly. The 362 clones were sequenced and BLAST analysis was conducted, 10 clones are shown to be novel ESTs, and were registered in GenBank. The mRNA levels of the seven novel genes were over expressed in LN686 cells treated by E1A protein compared to those of parental LN686 cells by semi quantitative RT PCR analysis, and the difference of mRNA expression was approximately 3 8 times. CONCLUSION:Ten novel gene fragments were isolated by the SSH technology, and it provided the basis for further cloning their full length genes a

关 键 词：肿瘤 化学治疗 耐药性 抑制性消减杂交 E1A蛋白 药敏相关基因 

分 类 号：R73-3[医药卫生—肿瘤]