论文部分内容阅读
目的通过研究乙型肝炎病毒(hepatitis B virus,HBV)长期感染与诱导性脱氨酶(activation induced deaminase,AID)表达水平的关系探讨肝癌发病机制。方法采用实时定量PCR法,测定乙肝病毒感染及转化生长因子β1(TGF-β1)和肿瘤坏死因子-α(TNF-α)对Apobec蛋白家族AID和Apobec3G表达量的影响;采用3D-PCR法检测AID、Apobec3G表达组对乙肝病毒基因突变的影响;克隆测序PCR扩增乙肝病毒X基因产物,对比AID和Apobec3G对乙肝病毒基因组突变的影响,计算突变率;并用逆转录病毒携带AID组和对照组在肝脏细胞转染表达后,克隆测序PCR细胞部分基因扩增,对比测定对细胞基因组突变的影响,计算突变率。结果乙肝病毒感染或生长因子TGF-β1刺激使肝细胞中AID表达上调>10倍,TGFβ1刺激组较非刺激组引起乙肝病毒基因组高突变,分别为55个克隆中的16和4个。AID高表达使乙肝病毒基因组高度突变,突变率为55个克隆中的16个,显著高于对照组的1个,多为G到A突变;而且AID高表达使部分细胞基因组如p53和c-myc基因高度突变,突变率分别为8.2×10-5和7.3×10-5,高于对照组的突变率0。结论慢性肝炎病毒感染协同细胞因子诱导AID高表达,高表达的AID使肝炎病毒基因及细胞基因组突变,为肝细胞癌变的相关因素。
OBJECTIVE: To investigate the pathogenesis of hepatocellular carcinoma (HCC) by studying the relationship between chronic hepatitis B virus (HBV) infection and the expression level of activation induced deaminase (AID). Methods The real-time PCR method was used to determine the effect of hepatitis B virus infection on the expression of Apobec protein AID and Apobec3G by using TGF-β1 and TNF-α. AID, Apobec3G expression group on hepatitis B virus gene mutations; cloning and sequencing PCR amplification of hepatitis B virus X gene products, compared AID and Apobec3G on the hepatitis B virus genome mutations, calculate the mutation rate; and with retrovirus carrying AID group and control group After transfection of liver cells, some genes of PCR cells were cloned and sequenced, and the effect on cell genome mutation was compared to calculate the mutation rate. Results Hepatitis B virus infection or TGF-β1 stimulation up-regulated the AID expression in liver cells by> 10-fold. The TGFβ1-stimulated group had higher mutations in the hepatitis B virus genome than the non-stimulated group, which was 16 and 4 out of 55, respectively. AID high expression of the hepatitis B virus genome was highly mutated, the mutation rate of 55 clones in 16, was significantly higher than the control group of 1, mostly G to A mutation; and AID high expression of some cell genomes such as p53 and c- The myc gene was highly mutated with a mutation rate of 8.2 × 10-5 and 7.3 × 10-5, respectively, which was higher than that of the control group. Conclusions Chronic hepatitis B virus infection synergizes with cytokines to induce high AID expression, and high expression of AID causes mutations in hepatitis virus genes and cell genomes, which are the related factors of hepatocellular carcinoma.