热休克蛋白90抑制乙型肝炎病毒复制的初步研究

Heat shock protein 90-mediated inhibition of hepatitis B virus replication in hepatic cells

目的了解热休克蛋白90（HSP90）对HBV复制的影响并探讨其可能的分子机制。方法构建人HSP90真核表达质粒pXF3H-HSP90（HA—HSP90），与HBV复制型质粒HBV1．3共转HepG2细胞，ELISA检测细胞上清液HBsAg表达水平，Southemblot检测HBV复制中间体的表达。将HA—HSP90表达质粒或HSP90siRNA转染HepG2细胞，实时定量逆转录聚合酶链反应检测白细胞介素（IL）-1D、IL-6以及干扰素应答基因1（IFIT1）的表达。将TANK结合激酶1（TBK1）siRNA、HBV1．3、HA—HSP90共染HepG2细胞，Southemblot检测HBV复制中间体的表达。多组间数据比较采用单因素方差分析，两两比较采用LSD检验。结果成功构建了表达HSP90的真核表达质粒HA—HSP90。转染HA—HSP90的HepG2细胞内高水平表达外源蛋白，而未转染质粒的细胞内没有检测到外源蛋白的表达。转染HA-HSP90质粒组细胞上清液中的HBsAg表达量吸光度值为0．124±0．033，明显低于转染空载体pXF3H组的0．340±0．011及未转染组的0．615±0．069（F=61．013，P〈0．05）。过表达HSP90也能抑制HBV复制，转染HA-HSP90组、转染空载体pXF3H组与未转染组HBV复制中间体表达量吸光度值分别为108．92±8．59、872．45±13．00和7856．37±60．23，差异有统计学意义（F=28260．00，P〈0．05）。在HepG2细胞内高表达HSP90能诱导高水平的IFIT1产生，其中HA—HSP90组与空载体对照pXF3H组IFIT1表达的2 ^△△ CT值分别为82．139±0．919和5．579±0．644，差异有统计学意义（F=13．108，P〈0．05），但未检测到IL-1β与IL-6的诱导表达。下调HepG2细胞中干扰素信号通路分子TBK1不影响HSP90对HBV的抑制，其中HSP90组与siTBK1±HSP90组HBV复制中间体条带灰度值分别为1952．64±67．88和2366．64±71．16（F=31．30，P〉0．05）。结论HSP90能够抑制HBV的复制与蛋白表达，这种抑制作用不依赖于TBK1通路，也与IL-1β信号通路无关。

Objective To evaluate the effect of heat shock protein 90 （HSP90） on hepatitis B virus （HBV） replication in hepatocytes and to investigate the related molecular mechanism. Methods A eukaryotic plasmid expressing human HSP90 was constructed （designated as HA-HSP90）. HepG2 cells were cotransfected with HA-HSP90 and the HBV replicative plasmid HBV1.3. Expression of the exogenous HSP90 was assessed by Western blotting. Expression of the HBV surface antigen （HBsAg） was determined by enzyme-linked immtmosorbent assay, and HBV replicative intermediates were detected by Southern blotting. Small interfering （si）RNAs were designed against HSP90 and TBK1 and transfected into the HepG2 cells to further assess the effects of HSP90 and its underlying mechanism. HSP90-mediated effects on the expression of interleukins IL-1β and IL-6 and the interferon response gene IFIT1 were assessed by quantitating mRNA levels with real time RT-PCR. Results The HA-HSPg0 plasmid successfully expressed exogenous HSP90 protein in HepG2 cells. The exogenous HSP90 was able to inhibit HBV replication and HBsAg expression. IFIT1 expression was up-regulated after HA-HSPg0 transfection, but neither IL-1β nor IL-6 were affected. The siRNA-mediated TBK1 down-regulation had no effect on the HSP90-inhibited HBV replication. Conclusion HSP90 can inhibit HBV replication and TBK1 is not involved in this process.