摘要目的 探索肝门部胆管癌组织内有无乙型肝炎病毒(HBV)基因的整合,并鉴定其在细胞染色体上的整合位点.方法 收集10例肝门部胆管癌新鲜组织标本,并以同期1例因十二指肠乳头癌而行胰十二指肠切除术患者的正常肝门部胆管组织标本作为对照.抽提组织DNA,采用HBV-Alu-PCR方法扩增HBV X基因及其侧翼的人基因组片段.PCR产物经序列测定后,应用序列分析软件分析比较HBV X基因序列,对其上、下游序列进行Entrez BLAST和NCBI MapViewer分析,确定HBV整合在染色体上的精确位置.结果 10例肝门部胆管癌组织中,有5例可检测到HBV整合片段,共获得6个整合位点.序列分析发现HBV X基因均以截断形式插入到宿主细胞基因组,其中CpG结合蛋白基因上游被HBV重复整合,与肿瘤发生密切相关的p53基因亦被HBV整合.结论 肝门部胆管癌组织内有较高的HBV整合率,HBV常整合在与肿瘤发生相关的基因中,HBV感染与肝门部胆管癌发生有关.

abstractsObjectives To study the phenomena of hepatitis B virus (HBV) integration into the tissues of hilar cholangiocarcinoma (HCCA) and to identify the integration sites in the host genome.Methods Ten fresh HCCA samples were collected from the tissues by surgical ablation, 1 normal hilar bile duct sample selected as control.Cellular DNA were extracted by Wizard(R) SV Genomic DNA Purification System.PCR-derived assay(HBV-Alu-PCR) was employed to amplify the viral-host junctions which contain the HBV sequence and the adjacent cellular flanking sequences.The PCR products were purified and subjected to sequencing by ABI-3730XL Auto DNA Analyzer.The sequence analysis of viral-host junctions was performed by DNASIS(R) MAX 3.0 bioinformatics software.The insertion sites between viral and cellular sequences were identified through homology comparison using NCBI BLAST and MapViewer search.Results In 10 HCCA samples,5 were demonstrated to have HBV integration fragments with total 6 inserted sites identified.Sequence analysis from viral-host junction showed that HBV X gene inserted into host genome at random distribution with truncated fragments.HBV integration recurrently targeted the unknown region in upstream of CXXC finger protein-1 (CpG-binding protein) gene (4 cases).p53 tumor suppressor gene was also found at the integration site.Conclusions There is high integration rate of HBV DNA into cellular genome of HCCA.HBV integration is found frequently into or close to cancer-related genes.The findings demonstrat that HBV infection might have association with the pathogenesis of HCCA.