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目的评价HBV G1613A和C1653T变异对乙型肝炎患者疾病进展、病毒体外复制力及核心启动子(CP)转录活性的影响。方法共纳入258例研究对象,包括65例急性乙型肝炎(AHB)患者,120例慢性乙型肝炎(CHB)患者和73例慢加急性肝衰竭(ACLF)患者。从患者血清中提取HBV DNA,PCR扩增HBV DNA全长基因组,统计G1613A、C 1 6 5 3 T和G 1 6 1 3 A+C 1 6 5 3 T变异的发生率。构建相应载体进行体外功能实验,观察病毒质粒转染He p G 2细胞后对病毒复制力及其CP转录活性的影响。结果 258例患者中共检出B、C、D三种基因型,其检出率分别是22.2%、76.2%和1.6%。G1613A、C1653T及G1613A+C1653T变异发生率随疾病程度加重依次升高。AHB、CHB和ACLF患者上述3种变异的检出率分别为13.70%、31.80%和45.20%(P<0.01),2.30%、16.30%和27.40%(P<0.01),2.29%、12.07%和23.29%(P<0.05)。与野生株相比,G1613A变异株复制力升高6%,HBs Ag降低15%,HBe Ag表达呈阴性,CP转录活性降低16.2%;C1653T变异株复制力升高10%,HBs Ag升高55%,HBe Ag与野生株接近,CP转录活性升高17.1%;G 1 6 1 3 A+C 1 6 5 3 T变异株复制力升高7%,H B s A g升高6 6%,H B e A g升高2 2 7%,但对CP转录活性没有影响。结论 G1613A、C1653T在CP区的变异可增加HBV复制力,影响CP转录活性和HBV抗原的表达,G1613A+C1653T联合变异可能对这些功能产生协同作用,推测这三种变异与乙肝重症化发生机制相关。
Objective To evaluate the effect of HBV G1613A and C1653T mutations on disease progression, replication in vitro and transcription activity of core promoter (CP) in hepatitis B patients. METHODS: A total of 258 study subjects were included, including 65 patients with acute hepatitis B (AHB), 120 patients with chronic hepatitis B (CHB) and 73 patients with acute and severe acute liver failure (ACLF). HBV DNA was extracted from patient serum and the full-length genome of HBV DNA was amplified by PCR. The incidence of G1613A, C1635T and G1631A + C1653T mutations were calculated. The corresponding vector was constructed for in vitro functional experiments to observe the effect of viral plasmid transfection on HepG2 cells and its replication activity and CP transcriptional activity. Results The genotypes B, C and D were detected in 258 patients. The detection rates were 22.2%, 76.2% and 1.6% respectively. The incidence of G1613A, C1653T and G1613A + C1653T mutation increased with the increase of disease severity. The detection rates of AHB, CHB and ACLF were 13.70%, 31.80% and 45.20% (P <0.01), 2.30%, 16.30% and 27.40% (P <0.01), 2.29% and 12.07% 23.29% (P <0.05). Compared with the wild type strain, the replicative power of G1613A mutant strain increased by 6%, the HBsAg decreased by 15%, the expression of HBeAg was negative and the CP transcriptional activity decreased by 16.2%. The replicative force of C1653T mutant strain increased by 10% and HBsAg increased by 55% %, HBe Ag was close to the wild-type strain, and the CP transcriptional activity increased by 17.1%. The replicative power of G 1 6 1 3 A + C 1 6 5 3 T mutant increased by 7%, HB s A g increased by 6 6% e A g increased by 2 2 7%, but had no effect on CP transcriptional activity. Conclusion Mutation of G1613A and C1653T in CP area can increase HBV replication and affect CP transcription activity and HBV antigen expression. G1613A + C1653T combined mutation may have a synergistic effect on these functions, suggesting that these three mutations are associated with the pathogenesis of severe hepatitis B .