乙型肝炎肝硬变患者外周血和肝组织中HBV cccDNA的定量检测  

Quantification of hepatitis B virus covalently closed circular DNA in peripheral blood and hepatic tissues of cirrhotic patients with hepatitis B

作　　者：李莹[1] 韩涛[1] 高英堂[1] 刘磊[1] 王毅军[1] 景丽[1] 张涛[1] 杜智[1] 

机构地区：[1]天津医科大学三中心临床学院天津市人工细胞重点实验室,天津市300170

出　　处：《世界华人消化杂志》2008年第33期3752-3758,共7页World Chinese Journal of Digestology

基　　金：天津市科委应用基础研究计划面上项目;No.06YFJMJC13100;天津市重点科技攻关专项资助项目;No.05YFSJSF02500;国家重点基础研究发展规划(973计划)资助项目;No.2007CB512801~~

摘　　要：目的:研究乙型肝炎肝硬变患者HBV共价闭合环状DNA(cccDNA)在肝组织和外周血中的分布及临床应用.方法:选取60例乙型肝炎肝硬变患者肝组织和外周血样本,外周血样本以不降解质粒的ATP依赖的DNA酶(PSAD)进行酶切,肝组织样本以限制性内切酶MluⅠ联合PSAD酶切而后进行荧光定量PCR检测.结果:60例乙型肝炎肝硬变患者外周血HBVcccDNA均阴性,肝组织HBV cccDNA阳性24例(40.00%),在HBeAg(+)组、HBeAg(-)HBeAb(-组、HBeAb(+)组分布分别为66.67%、52.94%和26.47%,定量结果在3组呈递减趋势,且HBeAg(+)组与HBeAb(+)组差异存在统计学意义(P<0.05).肝组织HBV cccDNA占总HBVDNA 0%-7.77%,肝组织HBV cccDNA与肝组织总HBV DNA存在相关性(r=0.53,P<0.01)与外周血总HBV DNA无相关性(r=0.18,P=0.18),与ALT、TBIL均无相关性(r=0.15,P=0.25;r=0.01,P=0.94).结论:乙型肝炎肝硬变患者外周血检测不到HBV cccDNA,肝组织HBV cccDNA占总HBVDNA比例较低,HBeAg(+)患者病毒复制最为活跃.AIM：To investigate the distribution of hepatitis B virus（HBV）covalently closed circular DNA（cccDNA）in peripheral blood and liver tissues of liver cirrhosis patient with hepatitis B virus infection.METHODS：DNA was extracted from serum andliver biopsy samples of 60 cirrhotic patients with hepatitis B virus infection.The serum samples were treated with Plasmid-Safe ATP dependent Dnase（PSAD）,while the liver biopsy samples were digested with restriction enzymes MluI and PSAD to reduce non-HBV cccDNA;then the total HBV DNA and cccDNA were measured by the real-time PCR assay.RESULTS：HBV cccDNA was negative in all the serum samples,but positive in 40%（24/60）he-patic biopsy samples.In HBeAg-positive,HBeAg-negative plus HBeAb-negative,and HBeAb-pos-itive hepatic tissues,HBV cccDNA positive rate was 66.7%,52.9%and 26.5%,respectively.Mean-while,the positive rate had significant difference between HBeAg-positive group and HBeAb-positive group（P〈0.05）.Intrahepatic cccDNA accounted for 0%-7.77%of total HBV DNA,and there was a significant correlation between intra-hepatic cccDNA and total HBV DNA（r=0.53,P 〈0.01）.Intrahepatic cccDNA had no correlation with the total HBV DNA in the serum samples,as well as with ALT and TBIL levels（r=0.15,P=0.25;r=0.01,P=0.94）.CONCLUSION：HBV cccDNA can not be detect-ed in peripheral blood of cirrhotic patients with hepatitis B virus infection.Intrahepatic cccDNA accounts for a small proportion of the total HBV DNA.Replication of HBV is the most active in HBeAg-positive patients.

关 键 词：肝硬变 乙型肝炎病毒 共价闭合环状DNA 实时荧光定量PCR 

分 类 号：R512.62[医药卫生—内科学] R361[医药卫生—临床医学]