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目的 研究干扰素 α(IFN α)抗乙型肝炎病毒(HBV)的信号转导机制。方法 用定量聚合酶链反应(PCR)检测IFNα2b处理前、后的HepG2 2 15细胞上清的乙型肝炎病毒脱氧核糖核酸(HBVDNA)水平。半定量逆转录聚合酶链反应(RT PCR)检测IFNα2b处理后不同时点的HepG2 2 15细胞和其母源细胞HepG2 细胞内信号转导和转录活化因子1(STAT1)、STAT2、干扰素刺激基因因子3γ(ISGF3γ)、2′5′寡腺苷酸合成酶(2′5′OAS)、RAN依赖蛋白激酶(PKR)的mRNA表达水平。WesternBlot检测ISGF3γ的蛋白质表达水平。并用染料木黄酮阻断IFNα2b信号转导后再次检测上述指标。结果 IFNα2b处理8h后,HepG2 2 15细胞上清HBVDNA平均减少0 72log 10copies ml,而加染料木黄酮后则无减少。IFNα2b处理后,HepG2 和HepG2 2 15细胞中STAT1、STAT2、ISGF3γ、2′5′OAS和PKRmRNA水平明显升高;加染料木黄酮后前3个因子mRNA水平仍然能检测到,而2′5′OAS和PKRmRNA则受到抑制。WesternBlot检测也提示IFNα2b处理后ISGF3γ蛋白质水平升高,加染料木黄酮后其表达受到抑制。结论 Janus酪氨酸激酶(JAK) STAT途径在IFN α抗HBV中发挥主要作用,而ISGF3是该途径的一个重要因子。
Objective To study the signal transduction mechanism of interferon α (IFN α) against hepatitis B virus (HBV). Methods Hepatitis B virus DNA (HBV DNA) levels in HepG2 2 15 cells before and after IFNα2b treatment were detected by quantitative polymerase chain reaction (PCR). Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the expression of STAT1, STAT2, interferon stimulating gene in HepG2 2 15 cells and their parental HepG2 cells at different time points after IFNα2b treatment Factor 3γ (ISGF3γ), 2’5 ’oligoadenylate synthetase (2’5’OAS), RAN-dependent protein kinase (PKR) mRNA expression levels. Western Blot detection ISGF3γ protein expression levels. And with genistein block IFNα2b signal transduction again after the above indicators. Results After treated with IFNα2b for 8 hours, the HBVDNA in HepG2 2 15 cells decreased by an average of 0 72 log 10 copies ml, while no decrease was observed after adding genistein. After treatment with IFNα2b, the levels of STAT1, STAT2, ISGF3γ, 2’5’OAS and PKR mRNA in HepG2 and HepG2 2 15 cells were significantly increased; the mRNA levels of the first three factors were still detected after adding genistein, OAS and PKR mRNA are inhibited. Western Blot also suggested that the level of ISGF3γ protein was increased after IFNα2b treatment, and the expression of ISGF3γ was inhibited after adding genistein. Conclusions Janus tyrosine kinase (JAK) STAT pathway plays a major role in IFNα anti-HBV, and ISGF3 is an important factor in this pathway.