肝癌中乙型肝炎病毒整合位点的研究  

Identification of hepatitis B virus integration sites in hepatocellular carcinoma tissues from patients with chronic hepatitis B

作　　者：屠红[1] 高海峰[2] 马国豪[2] 刘毅[2] 

机构地区：[1]上海市肿瘤研究所上海交通大学肿瘤研究所,200032 [2]复旦大学上海医学院

出　　处：《中华医学杂志》2006年第18期1249-1252,共4页National Medical Journal of China

基　　金：国家自然科学基金资助项目(30271455);"癌基因及相关基因国家重点实验室"开放课题基金资助项目(801)

摘　　要：确定HBV整合在染色体上的精确位置。结果在40例乙肝表面抗原阳性的肝癌组织中,有34例(85%)存在HBV整合现象。每份标本中的病毒整合拷贝数为1～5个不等,因此共获得了68个病毒整合位点。从病毒基因分析,整合可发生于X基因的任何长度,并不集中于通常认为的HBVDR1和DR2区,但在96%(65/68)的整合中,X基因均以截短形式插入宿主细胞DNA;从宿主基因分析,HBV偏好插入于基因的内含子和上游调控区,未见外显子中的插入。这些基因80%已被报道与肿瘤有关,它们的产物大多与细胞基本生存和死亡密切相关,可在各个细胞调控环节上促进肿瘤生成。十分有意义的是,在总共26个基因中即有3个基因(myeloid/lymphoidormixed lineageleukemia4,G proteinalphatransducingactivity polypeptide1和fibronectin1)发现被HBV重复整合。结论HBV在肝癌细胞染色体上的整合并不呈均衡分布,病毒对宿主细胞的“插入诱变”及整合子中持续表达的“截短型X蛋白”可能在病毒的致癌过程中起重要作用。Objective Hepatitis B virus （HBV） integration into the host genome is frequently detected in HBV positive hepatocellular carcinoma（HCC） in China. The aim of this study is to carry out a large-scale screening for the HBV integrations sites in HCC samples from Chinese patients. Methods Cellular DNA was extracted from 40 HBV-related HCC by proteinase K digestion/phenol extraction method. One primer specific to HBV sequence and another primer directed to human Alu repeat were used to amplify the virus/cellular DNA junction. To avoid undesirable amplifications between Alu sequences, primers were constructed with dUTPs and destroyed by uracil DNA glycosylase treatment after 15 initial cycles of amplification. Only desirable fragments were then further amplified with specific primers to the known region and to a tag sequence introduced in the Alu-Specific primer. The PCR product was purified and subject to direct sequencing by ABI 3700 Auto sequencer. NCBI （national center for biotechnology information ） BLAST and MapViewer search were used for identification of HBV location on human genomes. Results In 40 HBsAg positive HCC samples, 34（85% ） were showed to have at least one copy of HBV fragment in host genome, indicating HBV-Alu-PCR is a rapid way for identification of new cellular DNA sequences adjacent to HBV. Analysis from the 68 isolated viral-cellular junctions, X gene was found to be interrupted at any length, not specifically at DR1 and DR2 regions. Three-prime-deleted X gene was observed in 65 （96%） cases. HBV preferred to integrate into the intron and the up-stream regulatory region of the cellular genes. In no case HBV inserted into the exon. Our results also demonstrated that the cellular genes targeted by HBV are usually key regulators of cell proliferation and cell death. Three genes, myeloid/lymphoid or mixedlineage leukemia 4, G protein alpha transducing activity polypeptide 1 and fibronectin, were found to be recurrently targeted by HBV. Conclusion HBV-Alu-PCR is a powerful tool for

关 键 词：癌 肝细胞 肝炎病毒 乙型 病毒整合 

分 类 号：R735.7[医药卫生—肿瘤]