采用熔点曲线法研究乙肝病毒准种与拉米夫定临床耐药相关性

为了研究乙型肝炎病毒(HBV)准种与拉米夫定耐药的关系以指导临床用药,随机选取拉米夫定治疗后发生YMDD变异的慢性乙型肝炎(CHB)患者30例,以及停用拉米夫定后发生病毒反弹的CHB患者30例作为研究对象,同时以未经拉米夫定治疗的CHB患者30例为对照,采用聚合酶链反应(PCR)扩增这些病人体内HBV的P区,再用熔点曲线法分析3组患者体内的HBV准种情况。另外,采用相同方法对HBV C区、S区准种也进行了对比。结果显示,病毒变异组患者体内HBV P区准种数量为2.50±0.86个,病毒反弹组为5.30±0.95个,未治疗组为8.37±0.93个,3组间准种数量两两比较均有显著性差异(P<0.05)。HBV C区病毒变异组准种数量为6.10±1.86个,病毒反弹组为6.37±1.81个,未治疗组为6.33±1.64个,3组准种数量无显著性差异(P>0.05)。HBV S区病毒变异组准种数量为5.23±1.85个,病毒反弹组为6.17±1.93个,未治疗组为5.77±2.11个,3组准种数量无显著性差异(P>0.05)。HBV P区熔点曲线图显示,病毒变异组优势病毒群的熔点与病毒反弹组和未治疗组相比,已发生明显的偏移,而在HBV C区和S区熔点曲线图中,3组的波峰数和优势病毒群的熔点均没有明显变化。可见,在拉米夫定的作用下HBV P区准种数量减少,准种的性质也发生变化,发生变异后劣势耐药病毒株变为优势病毒株易被检测到。拉米夫定对HBV C区、S区作用不明显。

Reciprocity between Hepatitis B Virus Quasispecies to Clinical Tolerance of Lamivudine Adopting Melting Point Curve

Reciprocity between hepatitis B virus quasispecies and lamivudine tolerance was studied in order to guide clinieal rnedieation, 30 eases of the chronic hepatitis B （CHB） patients who were treated with lamivudine and had YMDD variation were seleeted randomly, 30 eases of CHB patients who had virus rebounding after stopping therapy with lamivudine, and 30 eases of CHB patients who had never have lamvudine therapy as control, were randomly se- lected. The HBV polymerase genes of these patients were amplified by PCR, then adopted melting point curve to analyze the quasispecies condition of HBV in three groups of patients. Moreover, the HBV C/S gene was analyzed by the same means for comparison with P gene. The results showed the quasispecies number of HBV P gene in virus variation group was 2.50 ±0. 86, that in virus rebounding group was 5.30 ±0.95, and that in without treatment group was 8.3± 0. 93. There were clear difference among three groups （ P 〈 0. 05 ）. The quasispecies number of HBV core gene in virus variation group was 6.10 ± 1.86, virus rebounding group 6.37 ± 1.81, and non-treatment group 6.33 ± 1.64 with no significant difference among the three groups （P 〉 0.05 ）. The quasispecies number of HBV S gene in virus variation group was 5.23 ± 1.85, rebounding group 6.17 ±1.93, and non - treatment group 5.77 ± 2.11 with no significant difference among three groups （P 〉 0.05 ）. Melting point curve of HBV P gene revealed that as compared the melting point of dominant group of virus variation group with virus rebound group and non - treatment group had happened apparent driftage. While on the melting point curve of HBV C and S gene, the number of wave crests of the three groups and the melting point of dominant virus group had no apparent change. Therefore, it was believed that under the effect of lamivudine the number of quasispecies of HBV P gene decreased and the feature of quasispecies had changed as well. After the variation, the inferior tolerant virus strain had changed into dominant virus strain and easily detectable. Lamivudine had no significant effect on HBV G and S genes.