荧光定量检测HBV cccDNA在慢性乙型肝炎患者中的应用

The application of cccDNA fluorescence quantitative detection of hepatitis B virus in patients with chronic hepatitis B

目的:研究慢性乙型肝炎患者HBV共价闭合环状DNA(cccDNA)在外周血的分布、肝组织中含量及探讨抗病毒治疗对慢性乙型肝炎患者血清HBV cccDNA的影响。方法:随机选取HBV DNA阳性慢性乙型肝炎患者、肝硬化患者、重型肝炎患者共125例。使用实时荧光定量PCR法检测血清中cccDNA。选取36例慢性乙型肝炎患者肝组织和外周血样本,酶切后进行荧光定量PCR检测。60例慢性乙型肝炎患者,按1:1随机分配接受拉米夫定或干扰素治疗,所有患者均治疗24周以上;应用实时荧光定量聚合酶链反应技术检测慢性乙型肝炎患者0周、8周、12周、24周、HBV cccDNA及HBV DNA含量。结果:125例患者中cccDNA阳性率为71.2%,以肝硬化患者阳性率最低。肝硬化患者HBVcccDNA检出阳性率与慢性乙型肝炎患者及重型肝炎患者比较,差异有显著性意义(P<0.05)。肝组织中HBV cccDNA与肝组织总HBV DNA及血清HBV DNA存在相关性(P<0.05),HBeAg阳性组与阴性组患者比较差异有显著性意义(P<0.05)。患者接受抗病毒治疗后血清HBV DNA及HBV cccDNA下降。结论:乙型肝炎肝硬化患者外周血HBV cccDNA阳性率低,慢性乙型肝炎患者中HBeAg(+)组病毒复制较HBeAg(-)组活跃。抗病毒治疗对血清HBV cccDNA有抑制作用;血清HBV cccDNA可作为抗病毒治疗的重要监测指标。

Objective：To study HBV covalently closed circular DNA （cccDNA） in the distribution of peripheral blood and liver tissue content in the patients of chronic HBV infections,investigate the impact of anti-viral treatment on serum HBV covalently closed circular DNA（cccDNA）of patients with chronic hepatitis B.Methods：Randomly selected a total of 125 cases with HBV DNA positive chronic hepatitis B,cirrhosis,severe hepatitis patients,using real-time fluorescence quantitative PCR for the detection of serum cccDNA.DNA were extracted from serum and liver biopsy samples of 36 patients with chronic hepatitis B,and were measured by the real-time fluorescence quantitative PCR assay.Sixty cases of chronic hepatitis B patients were randomly assigned to receive lamivudine or interferon treatment in a ratio of 1：1.Serum HBV cccDNA and HBV DNA levels were detected with real-time PCR at the time of 0,8,12,24 weeks.Results：The cccDNA-positive rate was 71.2% in 125 patients,to the lowest positive rate in patients with cirrhosis.The positive rate of HBV cccDNA had significant difference between patients with cirrhosis and chronic hepatitis B patients or severe hepatitis patients（P〈0.05）.There was a significant correlation between intra-hepatic cccDNA,intra-hepatic total HBV DNA and serum HBV DNA（P〈0.05）.Mean-while,the positive rate had significant difference between HBeAg-positive group and HBeAg-negative group（P〈0.05）.After receiving antiretroviral therapy,level of serum HBV DNA and HBV cccDNA was decreased.Conclusion：Positive rate of serum HBV cccDNA in patients with cirrhosis is low.Viral replication in HBeAg（＋） group is more active than in HBeAg（-） group.Anti-viral therapy has inhibition on serum HBV cccDNA;serum HBV cccDNA can be used as an important anti-viral treatment indicators for monitoring.