论文部分内容阅读
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
Objective To study the structure and function of a splicing variant of 2 2 kb hepatitis B virus in liver tissues of patients with liver cancer. Methods The whole genome of Hepatitis B virus (HBV) was amplified by PCR from 12 pairs of hepatocellular carcinoma and adjacent noncancerous liver tissues. The 3 2kb full-length HBV genome and the 2 2kb splice variant genome were cloned, sequenced and their differences in gene structure were compared. HepG2 cells were co-transfected with a 2 2 kb HBV splicing genome and a full-length genome. HBV full-length genome-specific primers and splice variant-specific primers were used to detect the intracellular HBV core particles after transfection to determine whether the 2 2 kb HBV splicing Effect of variants on full length HBV replication. Results 2 2 kbHBV genomic splice variants were found in all cancerous and paracancerous tissues. The same template amount of amplification products obtained by image scanning analysis showed that 2 2kbHBV genome splicing variants in cancerous tissue and full-length HBV ratio higher than adjacent tissues. Sequence analysis showed that the 2 2 kb HBV splice variant retained the 5 ’end of the packaging signal as well as the complete X, C and preC genes. The results of cell transfection showed that the replication of the 32kb full-length HBV genome could be increased by 3 ~ 7 times when transfected with 2 2kb splice variant. Conclusions There is a universal 25 kb splicing variant of HBV genome in liver tissue. The relative amount of this variant in cancerous tissue is higher than that in paracancerous tissues. The 2 2 kb HBV genomic splice variant can enhance the replication of the full-length HBV genome, suggesting that it may be related to the occurrence and development of hepatocellular carcinoma