【摘要】 目的　研究肝癌患者肝组织中 2 2kb乙型肝炎病毒基因组剪接变异体的结构和功能。方法　PCR扩增 12对肝癌组织及癌旁肝组织中的乙型肝炎病毒 (HepatitisBvirus,HBV)全基因组。克隆 3 2kb全长HBV基因组及 2 2kb剪接变异体基因组 ,测序并比较它们基因结构的差异。将 2 2kbHBV剪接体基因组与全长基因组共同转染HepG2 细胞 ,分别以HBV全长基因组特异性引物及剪接变异体特异性引物对转染后细胞内HBV核心颗粒进行PCR检测 ,以判定 2 2kbHBV剪接变异体对全长HBV复制功能的影响。结果　 2 2kbHBV基因组剪接变异体见于所有的癌组织及癌旁组织。相同模板量获得的扩增产物进行图象扫描分析 ,发现癌组织中 2 2kbHBV基因组剪接变异体与全长HBV的比值高于癌旁组织。序列分析表明 ,2 2kbHBV剪接变异体保留 5′端包装信号以及与完整的X基因、C及preC基因。细胞转染结果显示 ,加入 2 2kb剪接变异体共转染 ,细胞中 3 2kb全长HBV基因组的复制量可增强 3～ 7倍。结论　肝组织内普遍存在 2 2kbHBV基因组剪接变异体 ,该变异体在癌组织中的相对量高于癌旁组织。 2 2kbHBV基因组剪接变异体可使全长HBV基因组复制增强 ,提示可能与肝癌的发生、发展相关更多还原

【Abstract】 Objective To study the structure and function of 2.2 kb spliced variant of HBV genome from liver tissues of hepatocellular carcinoma patients.Methods HBV genomes were amplified by using PCR from paired hepatocellular carcinoma tissues and peritumor tissues.The 3.2 kb full-length HBV genome and 2.2 kb spliced variant were separately cloned and sequenced.Hep G 2 cells were co-transfected with full-length HBV DNA and 2.2kb spliced variant,and after transfection,HBV DNAs from intracellular core particles were harvested and specific primers were used in PCR to evaluate the interactions between spliced variant and full-length counterpart in replication.Results Semi-quantification by scanning density showed that 2.2 kb spliced variant was present in all tumor and peri-tumor samples studied.Sequence analyes revealed that the 5′ terminus packaging signal for pregenomic and X and PreC/C genes were retained.When full-length HBV DNA was co-transfected with 2.2 kb,the replication signal of 3.2 kb HBV genome was increased 3～7 times.Conclusion The 2.2 kb HBV spliced variant was present in liver tissues,and relative content was higher in tumor tissues than that in the peri-tumor tissues.This spliced variant could enhance the replication of full-length HBV genome,which suggested the possible role of the variant in the pathogenesis of development of hepatocellular carcinoma.更多还原