肝细胞核因子4α增强型1.0倍HBV复制子体外复制模型建立及初步应用  

Construction and initial application of HNF4 α enhanced 1.0-fold HBV replication model in vitro

作　　者：丁宁[1] 张明香[1] 

机构地区：[1]沈阳市第六人民医院肝病科,110006

出　　处：《肝脏》2016年第1期28-33,共6页Chinese Hepatology

基　　金：国家传染病重大专项分课题:慢性病毒性肝炎中西医结合治疗方案优化研究(2012ZX1005004-001)

摘　　要：目的建立一种肝细胞核因子4α（HNF4α）增强型1.0倍HBV复制子体外复制模型。方法从临床获得的急性乙型肝炎患者血清中扩增并克隆HBV全长DNA;手术获得肝组织中提取总RNA,扩增并克隆HNF4α基因cDNA。BspQI/ScaI双酶切回收HBV全长DNA,HNF4α基因cDNA定向连接到真核表达载体pcDNA3.1（＋）中,构建pcDNA3.1-4α表达载体。将HBV全长DNA1μg/孔,按比例共转染pcDNA3.1-4α（0,0.5μg,1μg）于HepG2细胞,96 h后检测细胞上清中的HBsAg、HBeAg和HBV DNA;以0.5μg/1μg pcDNA3.1-4α与HBV全长DNA共转染HepG2细胞,加入不同浓度LAM（0,100μmol/L）和ETV（0,10μmol/L）,评价建立的体外复制模型。结果 1%TAE琼脂糖凝胶电泳鉴定HBV全长DNA和HNF4αcDNA PCR产物,条带长度为3.2 kb和1.5 kb;目的片段克隆后测序鉴定正确。胶回收HBV全长DNA克隆质粒酶切目的片段,构建的pcDNA3.1-4α表达载体酶切鉴定正确后,测序鉴定。不同浓度比例的pcDNA3.1-4α与HBV全长DNA转染HepG2细胞后,0μg/1μg组合：HBeAg阴性,HBsAg阴性,上清HBV DNA载量为103;0.5μg/1μg组合：HBeAg阴性,HBsAg A值从0.1升高到1.99,上清HBV DNA载量从1×10^3 IU/mL升高到4×10^4 IU/mL;1μg/1μg组合：HBeAg阴性,HBsAg A值从0.1升高到2.4,上清HBV DNA载量从1×10^3 IU/mL升高到1×10~5 IU/mL;LAM从0~100μmol/L,细胞上清HBV DNA降低了97.6%;ETV从0~10μmol/L,上清HBV DNA降低了99.0%。结论 HNF4α增强型1.0倍HBV复制子体外复制模型,可以体外评价临床常用抗病毒药物,为后续HBV研究提供新思路。Objective To construct a hepatocyte nuclear factor 4 alpha（HNF4α）enhanced 1.0-fold hepatitis B virus（HBV）replication model.Methods A full-length HBV DNA from serum of acute hepatitis B（AHB）patients was isolated for amplification and cloning,and total RNA of liver tissue from surgery was extracted for HNF4 α gene cDNA amplifying and cloning.BspQI/ScaI was used to digest HBV DNA plasmid and recover HBV DNA production.HNF4 α gene cDNA was directionally connected to the eukaryotic expression vector pcDNA3.1（＋） for construction of pcDNA3.1-4 αexpression vector.HepG2 cells were transfected with full-length HBV DNA（1μg/well）,according to proportion of transfection pcDNA3.1-4α（0μg,0.5μg,1μg）.After 96 hours,HBsAg,HBeAg levels and HBV DNA load in cell supernatant were detected.HepG2 cells were transfected with 0.5μg/1μg pcDNA 3.1-4 α and full-length HBV DNA,which were added with different concentrations of LAM（0μmol/L,100μmol/L）and ETV（0μmol/L,10μmol/L）to evaluate the replication model in vitro.Results The 1% TAE agarose gel detection showed that the lengths of the strips of full-length HBV DNA and HNF4αcDNA PCR product were 3.2 kb and 1.5 kb,respectively,of which cloning target fragments were sequenced and identified.The full-length HBV DNA was recycled from gel for cloning digested target fragments.The constructed pcDNA3.1-4αexpression vector was sequenced and identified.HepG2 cells were transfected with different ratios of pcDNA 3.1-4 α and full-length HBV DNA.In 0μg/1μg group,HBeAg and HBsAg were negative with a HBV DNA load of 103 in supernatant;in 0.5μg/1μg group,HBeAg was negative and HBsAg OD rose from0.1 to 1.99 with an increasing HBV DNA load from1×10^3 to 4×10^4;in 1μg/1μg group,HBeAg was negative and HBsAg OD rose from 0.1 to 2.4 with an increasing HBV DNA load from 1×10^3 to 1×10~5.HBV DNA load in supernatant was reduced by 97.6% as lamivudine increasing from 0μmol/L to 100μmol/L,and was also reduced by 99.0% as entecavir increasing from 0μmol/L

关 键 词：HNF4α 乙型肝炎 体外复制 抗乙肝药物 

分 类 号：R512.62[医药卫生—内科学] R-332[医药卫生—临床医学]