四环素调控表达肿瘤坏死因子α抗乙型肝炎病毒的实验研究

摘要

目的 研究四环素调控表达的肿瘤坏死因子α (TNFα)抑制HBV复制的作用. 方法 以pcDNA4TM/四环素操纵子序列(TO)载体为模板,用PCR法扩增含四环素调控表达序列片段TO,插入pUC118载体中测序,选择测序正确的序列取代pVITRO3载体中相应的片段,构建成pVITRO3-TO.用相同的方法以pcDNA6/四环素阻遏蛋白(TR)(C)载体为模板扩增TR基因,经测序证实后插入pVITRO3-TO载体的一个多克隆位点,构建成pVITRO3-TO-TR.在另一个多克隆位点插入TNF α 基因,构建pVITRO3-TO-TR-TNF α .将重组质粒瞬时转染HepG2细胞,研究四环素调控表达TNF α 的有效性.以HepG2.2.15细胞为HBV复制模型,转染pVITRO3-TO-TR-TNF α 后,经潮霉素抗性筛选及有限稀释,选择高效表达TNF α 的细胞建立细胞系,观察经四环素调控表达后,TNF α 抑制HBV复制的作用.多组间数据比较使用方差分析. 结果 经过改建的双表达载体能够有效表达TNF α,并可以用不同剂量的四环素进行调控,四环素的最佳工作浓度为1.0μ g/ml.诱导1、3、5d后,转染pVITRO3-TOTR-TNF α 组上清液中的HBsAg抑制率分别为14.8％、11.5％、28.44％,转染pVITRO3-TOTR组分别为1.2％、1.1％、0,诱导5d后的HBsAg抑制率差异有统计学意义(F=13.65,P＜0.05);转染pVITRO3-TOTR-TNF α 组的HBeAg的抑制率分别为50％、26.67％、47.85％,转染pVITRO3-TOTR组分别为0.3％、1.3％、0,诱导1、3、5d时的HBeAg抑制率差异均有统计学意义(F值分别为27.31、14.06和43.76,P值均＜0.05).荧光定量PCR结果显示,诱导5d后,TNF α 对细胞内和培养上清液的HBV DNA有明显的抑制作用,抑制率分别为70.26％和79.86％,与对照组相比,差异均有统计学意义(F值分别为96.7和62.72,P＜0.05).进一步的研究结果表明,TNF α对HBV S基因的mRNA转录有明显抑制作用.结论 成功构建了四环素调控表达的TNF α 载体,瞬时和稳定转染细胞后均能有效表达.在细胞模型中对HBV DNA的抑制作用最好,其次是对HBeAg的抑制,对HBsAg的抑制作用最弱.TNF α对病毒转录水平的抑制可能是其发挥抗HBV作用的一个重要环节.%Objective To study the role of tumor necrosis factor-alpha (TNFα) in the anti-replication effects of tetracycline (Tet) on hepatitis B virus (HBV).Methods The Tet-dependent regulatory fragment (TO) was PCR amplified from the pcDNA4TM/TO vector,inserted into the pUC 118 cloning vector,and verified by sequencing.The counterpart fragment in the pVITRO3 expression vector,which contains two multiple cloning sites (MCSs),was replaced with the confirmed TO to generate a pVITRO3-TO vector.The Tet repressor (TR) gene from the PeDNA6/TR regulatory vector was incorporated into one MCS of pVITRO3-TO and the TNFα gene was subsequently incorporated into the other MCS.The resultant vector,pVITRO3-TOTR-TNFα,was transiently transfected into HepG2 cells.TNFα expression from the vector was induced by exposure to various concentrations of Tet and measured by enzyme-linked immunosorbent assay to determine the appropriate Tet concentration for experimentation.To investigate whether Tet inhibits TNFα expression as a mechanism of its anti-replication activity against HBV,the HepG2.2.15 cell line stably transfected with pVITRO3-TOTR-TNFα was used as an HBV replication model.Levels of hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) were detected by immunoassay.HBV DNA level was detected by fluorescence quantitative PCR.Results The TNFα expression from the newly constructed pVITRO3-TOTR-TNFα vector was Tet-controllable in the eukaryotic cells examined.The optimal concentration of Tet for the experimental system was 1.0 μg/ml.HBsAg and HBeAg expression was down-regulated in the HepG2.2.15 cells stably transfected with the pVITRO3-TO-TR-TNFα vector.After incubation with Tet for 1,3 and 5 days,the inhibition rate of HBsAg was 2％,1.1％ and 0,compared to 14.8％,11.5％ and 28.4％ in the non-Tet control group.The corresponding inhibition rates of HBeAg were 50.0％,26.7％ and 47.9％,compared to 0.3％,1.6％ and 0.0％,in the control group.HBV DNA levels in the cells and the cell culture supematants exposed to Tet were decreased by 70.3％ and 79.9％,respectively.TNFα inhibited production of HBsAg mRNA.Conclusion A Tet-dependent regulatory fragment double-expressing TNFα single vector system was constructed successfully,achieving controllable TNFα expression in both transiently transfected eukaryotic cells and stable cell lines.In this HBV cell model system,Tet-induced overexpression of human TNFα inhibited HBV DNA replication and reduced HBsAg and HBeAg expression.Inhibition of HBV transcription may be a key role of TNFα against HBV replication.