目的：研究人miR-155对HepG2.2.15细胞中乙型肝炎病毒（HBV）的抑制作用，为 miRNAs治疗乙型肝炎提供理论依据。方法以 HepG2.2.15细胞基因组为模板，PCR扩增人 miR-155前体序列，双酶切连接到 pmR-mCherry质粒，构建 pmiR -155真核过表达载体，并转染到 HepG2.2.15细胞。设重组组（ pmiR -155质粒）、空载组（ pmR -mCherry 质粒）、转染试剂组和空白组。转染后24 h、48 h、72 h，应用实时荧光定量PCR法检测各组 miR-155表达量和 HBV DNA拷贝数，化学发光法定量检测 HBsAg和 HBeAg变化情况。结果实时荧光定量 PCR结果显示，与空白组相比，重组组 miR -155表达量明显提高。化学发光法结果示在转染后48 h，过表达的 miR -155对上清培养液所分泌 HBsAg、HBeAg 抑制作用明显，抑制率分别为（83.96±1.52）%和（79.60±8.71）%。定量PCR检测示过表达的miR-155对HBV DNA拷贝数的抑制率分别为（51.87±0.36）%、（43.67±1.51）%和（68.21±6.02）%。结论 miR -155对 HBV 蛋白的抑制作用具特异性，呈负相关，在体外可抑制 HepG2.2.15细胞中 HBV的复制和表达。%Objective It is to investigate the depressant effect of microRNA -155(miR -155)on Hepatits B virus in HepG2. 2. 15 cell,and provide a theoretical basis for the treatment of hepatitis B. Methods The pre-miR-155 was amplified from total DNA of HepG2. 2. 14 by PCR. The target gene fragment was digested and cloned into the pmR-mCherry plasmid. The pmiR-155 was transfected into HepG2. 2. 15 cells by liposome-mediated method. The empty plasmid,the reagent group and untreated cells were set as control. The expression of miR -155 was detected by the real-time quantitive PCR. The ex-pression of HBsAg and HBeAg were detected by chemiluminescent assay,and the changes of HBV DNA were investigated by real-time quantitive PCR. Results MiR-155 level of HepG2. 2. 15 cells which were transfected with the recombinant plasmid was obviously higher than controls. The chemiluminescent assay showed that over-expression of miR-155 could inhibit the ex-pression of HBsAg and HBeAg with(69. 43 ± 0. 7)%,(45. 89 ± 0. 74)% respectively on the 2nd day post-transfection. The HBV DNA was markedly suppressed with(51. 87 ± 3. 74)%,(43. 67 ± 0. 687)% and(68. 21 ± 0. 47)% by real-time quantitive PCR. Conclusion The inhibition effect of HBV protein was specific and had a certain dependency on the expression of miR-155. MiR-155 can suppress the replication and expression of HBV in vitro.
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