拉米夫定与HBV YMDD耐药变异及临床相关因素分析

Detection of Lamivudine-resistant HBV YMDD Mutants and Investigation of Factors related to Development of HBV YMDD Mutants

目的:用PCR-RFLP方法检测慢性乙型肝炎患者HBV-YMDD变异发生的情况,并分析与YMDD变异发生有关的因素。方法:从慢性乙型肝炎患者血清中提取的HBV DNA,扩增HBV多聚酶YMDD主型区核苷酸序列,用针对突变位点的特异限制性内切酶酶切扩增产物,经6％聚丙烯酰胺凝胶电泳后用限制性片段长度多态性技术鉴定HBVYMDD变异。结果:在152例慢性乙型肝炎患者中,72例单用拉米夫定治疗、41例接受拉米夫定与干扰素联合治疗、39例未接受拉米夫定治疗的患者,HBV YMDD变异检出率分别是47.2％、19.5％、7.7％;72例单用拉米夫定治疗的患者,36例治疗2周～6个月、20例治疗7～12个月、16例治疗13～27个月,HBV YMDD变异检出率分别是25％、55％、87.5％。结论:在使用拉米夫定治疗慢性乙型肝炎的过程中可发生YMDD耐药变异,且随拉米夫定治疗时间的延长,变异发生率增加,但拉米夫定与干扰素联合进行抗病毒治疗可延迟或阻滞YMDD变异的发生。HBVYMDD变异可能自然存在或发生。HBV基因组其他位点的变异也可能使HBV对拉米夫定产生耐药性。

Objective: To detect Lamivudine-resistant HBV YMDD mutants in CHB patients treated with Lamivudine and investigate factors related to development of HBV YMDD mutants. Methods: HBV DNA was drawn from serum of CHB patients, amplified the 300-790 nucleoside of HBV DNA polymerase gene which include YMDD motif, cleaved PCR products with restriction endonuclease which is specific to mutation site, cleaved fragments were analysed by 6 % polypropylene acidemide gel electrophoresis and HBV YMDD mutants were identified by PCR restriction fragment length polymorphism. Results: Of 72 cases who received Lamivudine monotherapy, 41 cases who received combination therapy with Lamivudine and IFN-α and 39 cases who were not treated with Lamivudine, 34 cases, 8 cases and 3 cases were observed with HBV YMDD mutants respectively. Furtherly, of 72 cases treated with only Lamivudine, the periods of Lamivudine monotherapy in 36 cases treated with only Lamivudine, the periods of Lamivudine monotherapy in 36 cases, 20 cases and 16 cases was from 2 weeks to 6 months, over 6 months to 12 months and over 12 months to 24 months respectively, the cases who were observed with HBV YMDD mutants were 9 cases, 11 cases and 14 cases respectively. Conclusion: Therapy with Lamivudine may not only result in the development of HBV YMDD mutants, but prolonged Lamivudine therapy would lead to incidence of HBV YMDD mutants to increase, while combination therapy with Lamivudine and IFN -α would delay the development of HBV YMDD mutants. HBV YMDD mutants may exist before Lamivudine therapy or occur naturally. Mutation outside the YMDD motif may also contribute to clinical viral resisatnce to Lamivudine.