利用半肝切除再生模型研究单个肝细胞核内耐药鸭乙型肝炎病毒DNA的动态变化

Dynamic changes in drug-resistant duck hepatitis B virus DNA in single hepatocyte nucleus after hemihepatectomy using a liver regeneration model

目的利用半肝切除再生模型研究单个肝细胞核内耐药鸭乙型肝炎病毒(DHBV)DNA的动态变化规律。方法 45日龄慢性DHBV感染鸭半肝切除后接种突变株转染上清,持续饲喂拉米夫定,定期采血及取肝组织标本,检测外周血病毒DNA水平及病毒株,流式细胞仪分选单个肝细胞核及增殖细胞核抗原(PCNA)阳性与阴性细胞核,巢式PCR扩增核内DHBV YMDD区,直接测序法检测是否存在突变。计数资料组间比较采用χ~2检验和Fisher精确概率法。半肝切除前后PCNA的比较使用方差分析Welch法。结果半肝切除前单个核内病毒DNA及血清中克隆均为野生株;接种突变株转染上清后,在半肝切除术后1周时突变株感染的肝细胞核比率高于术后12周时的水平(χ~2=7.225,P<0.01);术前PCNA阳性肝细胞核比率为(0.84±0.36)%,术后1周时显著升至(42.26±6.48)%,术后12周时为(11.83±3.97)%,各时间点比较差异有统计学意义(F=190.832,P<0.001);术后1周时突变株感染的PCNA阳性细胞核比率高于术后12周时的水平,两组比较差异有统计学意义(χ~2=7.063,P<0.01),而术后PCNA阴性的细胞核突变株感染的比率极低。结论半肝切除可为耐药病毒提供复制空间,但耐药病毒在外周血及肝内未能形成优势毒株。

Objective To investigate the dynamic changes in drug-resistant duck hepatitis B virus（DHBV） DNA in single hepatocyte nucleus after hemihepatectomy using a liver regeneration model.Methods Ducks aged 45 days with chronic DHBV infection were inoculated with mutant DHBV after hemihepatectomy and then fed with lamivudine.Blood samples and liver tissue samples were collected at regular intervals.Viral DNA level in peripheral blood and viral strains were measured.Flow cytometry was used to isolate single hepatocyte nuclei and nuclei with or without proliferating cell nuclear antigen（PCNA）.Nested-PCR was used for the multiplication of intranuclear DHBV YMDD region.Direct sequencing was used to determine the absence or presence of mutation.The chi-square test and the Fisher＇s exact text were used for comparison of categorical data between groups.The Welch method was used for comparison of PCNA before and after hemihepatectomy.Results There were wild strains in single hepatocyte nuclei and serum before hemihepatectomy.After inoculation with mutant strains,the percentage of hepatocyte nuclei infected with mutant strains decreased significantly from 1 week to 12 weeks after hemihepatectomy（χ^2=7.225,P〈0.01）.The percentage of PCNA-positive hepatocyte nuclei was 0.84%±0.36% before surgery,significantly increased to42.26%±6.48% at 1 week after surgery,and was 11.83%±3.97% at 12 weeks after surgery,and there was a significant difference between different time points（F=190.832,P〈0.001）.The percentage of PCNA-positive nuclei infected with mutant strains decreased significantly from 1 week to 12 weeks after surgery（χ^2=7.063,P〈0.01）,while the percentage of PCNA-negative nuclei infected with mutant stains was extremely low after surgery.Conclusion Hemihepatectomy may provide a replication space for drug-resistant virus,but drug-resistant virus cannot become dominant strains in the peripheral blood and the liver.