拉米夫定与乙型肝炎病毒准种及变异特点的关系

Quasispecies and mutation of hepatitis B virus polymerase gene in lamivudine- treated patients

目的 研究拉米夫定治疗前后慢性乙型肝炎患者体内乙型肝炎病毒聚合酶(HBV P)基因序列的准种组成及变异特点。 方法 目的片段经聚合酶链反应扩增后克隆,每份标本选择3 3个克隆,用单链构象多态性/异源双链分析法对准种复杂性及变异特点进行分析。 结果 拉米夫定治疗前患者体内HBV P基因序列准种数为7～14(平均9.8),高于治疗后准种数4～8(平均5.7),t=3.98,P<0.05。6例患者治疗前的准种分布中有一种或两种优势准种,但比例较低(33.3％～81.8％);治疗后显著升高(78.8％～90.9％),t=3.42,P<0.05。选择优势克隆测序,6例患者经拉米夫定治疗后2例出现M550V/L526M变异,3例为M550I变异,1例无YMDD变异。此外为个体化的点突变,无明显趋势。 结论在拉米夫定的药物筛选作用下HBV准种的组成改变,同时出现YMDD序列变异。

Objective To study the quasispecies and mutation features of hepatitis B virus polymerase (HBV P) gene in chronic hepatitis B patients before and after lamivudine treatment. Methods The HBV P gene was amplificated with PCR and cloned, then single strand conformation polymorphism / heteroduplex analysis (SSCP/HDA) was applied to analyze the quasispecies complexity and mutation characters of HBV P gene. Results The quasispecies number of HBV P gene before treatment was larger than that after treatment (7-14 vs 4-8, t = 3.98, P < 0.05). Six patients had one or two predominant quasispecies before therapy, but the percentages of predominant quasispecies were lower than those after therapy (33.3%-81.8% vs 78.8%-90.9%, t = 3.42, P < 0.05). By sequencing the predominant clones, there were 2 patients with M550V/L528M mutation, 3 patients with M550I mutation and 1 patient with wild type after lamivudine therapy. Additionally, there were individualization mutations which had no obvious tender. Conclusion Quasispecies of hepatitis B virus are changed under the selection of lamivudine, meanwhile, the mutation at YMDD motif emerges.