摘要
目的探讨磁性纳米富集DNA及滚环扩增技术(RCA)检测外周血单个核细胞(PBMC)乙型肝炎病毒(HBV)共价闭合环状DNA(cccDNA)水平及其临床价值。方法选择西南医科大学附属医院98例HBV感染者为HBV组,另选择该院同期健康体检者100例为对照组。通过磁性纳米富集技术提取,RCA扩增、跨缺口引物介导的实时荧光定量PCR测定PBMC内HBV cccDNA;同时检测HBV DNA、HBsAg、HBeAg水平,取肝脏组织活检行炎症活动度、纤维化分期程度分级,并进行相关统计学分析。结果基于磁性纳米富集、RCA的检测技术能够测定PBMC内HBV cccDNA。HBV患者PBMC内HBV cccDNA阳性率为62.24%(61/98),伴肝衰竭、肝硬化患者PBMC内HBV cccDNA阳性率明显高于急性HBV、慢性HBV感染患者(P<0.05)。≥G2/S2期患者PBMC内HBV cccDNA量明显高于<G2/S2患者(P=0.001),血清HBV DNA≥1×105 copies/mL患者PBMC内HBV cccDNA水平明显高于<1×105 copies/mL患者(P=0.001)。PBMC内HBV cccDNA量与血清HBV DNA、HBsAg、HBeAg(HBeAg阳性患者)水平呈明显正相关(r=0.437、0.338、0.521,P=0.000、0.001、0.001)。抗病毒治疗24周[曲线下面积(AUC)=0.771,P=0.005]、48周(AUC=0.868,P=0.000)时PBMC内HBV cccDNA水平可对病毒应答明显预测。结论通过磁性纳米富集、RCA技术能够检测PBMC内HBV cccDNA,其水平与HBV疾病进展、血清感染标志物有一定关系,能够预测抗病毒治疗效果。
Objective To detect the level of hepatitis B virus (HBV) cccDNA in peripheral blood mononuclear cells (PBMC) by magnetic nanoparticle enrichment DNA and rolling circle amplification (RCA) and analyze its clinical value. Methods A total of 98 patients with HBV in the Affiliated Hospital of Southwest Medical University were selected as the subjects (group HBV),and the other 100 healthy controls were negative controls.The HBV cccDNA of PBMC were detected by magnetic nano enrichment DNA,RCA and real time fluorescence quantitative PCR by cross notched primers.The levels of HBV DNA,HBsAg and HBeAg were detected.Inflammatory activity and fibrosis stage classification were performed by liver biopsy.Data were statistical analyzed. Results The methods based on magnetic nano enrichment and RCA detection technology can determine the HBV cccDNA in PBMC.The positive rate of HBV cccDNA in PBMC was 62.24%(61/98) in HBV patients.The positive rate of HBV cccDNA in PBMC with liver failure and liver cirrhosis were significantly higher than that in acute HBV and chronic HBV patients ( P <0.05).The HBV cccDNA in PBMC of patients with ≥G 2/S 2 stage significantly higher than patients with <G 2/S 2 stage( P =0.001),the HBV cccDNA in PBMC of patients with HBV DNA ≥ 1× 10^5 copies/mL was significantly higher than patients with < 1× 10^5 copies/mL( P = 0.001 ).The level of HBV cccDNA in PBMC was positively correlated with serum HBV DNA,HBsAg and HBeAg (HBeAg positive patients) levels( r =0.437, P =0.000;r =0.338, P = 0.001;r = 0.521 , P = 0.001).At the 24th week (AUC=0.771, P =0.005) and 48th week (AUC= 0.868 , P = 0.000 ) after antiviral treatment,the HBV cccDNA level in PBMC could obviously predict the viral response. Conclusion The HBV cccDNA in PBMC can be detected by magnetic nano enrichment and RCA technique,and its content were related to the progression of HBV disease and serum infection markers.It could predict the effect of antiviral therapy.
作者
王友强
郭永灿
兰由玉
魏聪
刘鹏
WANG Youqiang;GUO Yongcan;LAN Youyu;WEI Cong;LIU Peng(College of Integrated Traditional Chinese and Western Medicine,Southwest MedicalUniversity,Luzhou,Sichuan 646000,China;Clinical laboratory,the AffiliatedTraditional Chinese Medical Hospital of Southwest Medical University,Luzhou,Sichuan 646000,China;Department of Rheumatology and Immunology,the Affiliated Hospitalof Southwest Medical University,Luzhou,Sichuan 646000,China;Department ofHepatobiliary Diseases,the Affiliated Traditional Chinese Medicine Hospital ofSouthwest Medical University,Luzhou,Sichuan 646000,China)
出处
《重庆医学》
CAS
2019年第9期1462-1466,1471,共6页
Chongqing medicine
基金
四川省卫生和计划生育委员会科研课题重点项目(16ZD0036)
西南医科大学-西南医科大学附属中医院联合专项项目(2016-001203001)
