干扰素α抗病毒活性的实验研究

目的比较不同亚型干扰素α（IFNα2b、IFNα2a、IFNαIb）对JAK-信号转导活化转录因子（STAT）通道中重要信号传导分子STAT1、STAT2、干扰素α受体（IFNAR）、蛋白激酶（PKR）、核糖核酸酶L（RNaseL）表达的影响，研究其抗病毒活性的差别，并评价IFNα抗病毒活性的关键性信号传导分子。方法1000U／ml的不同亚型IFNα分别作用于HepG2细胞后，用逆转录聚合酶链反应（RTPCR）方法检测HepG2细胞内STAT1、STAT2、IFNAR、PKR、RNase L mRNA表达水平。用western blot方法检测细胞内STAT1及IFNAR蛋白表达水平。结果RT-PCR的实验结果：（1）IFNα1b处理组IFNAR、STAT1、STAT2 mRNA的表达水平较IFNα2b组高，两组比较差异无统计学意义。IFNα1b、IFNα2b处理组IFNAR、STAT1、STAT2 mRNA水平明显较IFNα2a组高，差异均有统计学意义，，值分别为5．26、15．6、4．67，P值均〈0．05。（2）IFNα1b、IFNα2b、IFNα2a处理后的PKR表达水平相似，3组比较差异无统计学意义。（3）RNaseL表达量极少，无法比较不同处理组间RNase L mRNA表达水平的差异性。Western blot实验结果：（1）IFNα1b处理组IFNAR、STAT1蛋白水平较IFNα2b处理组高，两者比较差异无统计学意义，IFNα1b、IFNα2b处理组IFNAR、STAT1蛋白水平较IFNα2a处理组明显升高，差异有统计学意义，F值分别为17．7、20．1，F值均〈0．05。结论IFNα2b、IFNα2a、IFNα1b均可促进JAK—STAT信号通道中STAT1、STAT2、IFNAR mRNA及蛋白表达，IFNα1b和IFNα2b作用较强，IFNα2a作用较弱。初步表明，IFNα1b、IFNα2b的抗病毒活性较IFNα2a强，IFNAR、STAT1、STAT2可作为评价IFNα抗病毒活性的关键性指标，而PKR、RNaseL能否作为评价指标有待进一步的实验证实。

An experimental study on antiviral effects of IFN α

OBJECTIVE: To investigate the effects of different subtypes IFN alpha (IFN alpha2b, IFN alpha2a, and IFN alpha1b) transduction molecular STAT1, STAT2, IFNAR, PKR, and RNase L, and to study the differences of their antiviral effects and to evaluate the key signaling transduction molecules. METHODS: (1) After HepG2 cells were treated with IFN alpha2b, IFN alpha2a, or IFN alpha1b, the mRNA levels of STAT1, STAT2, IFNAR, PKR, and RNase L were detected by RT-PCR. (2) After HepG2 cells were treated with 1000 U/ml IFN alpha2b, IFN alpha2a, or IFN alpha1b, the protein expression levels of STAT1 and IFNAR were examined by Western blot. RESULTS: RT-PCR results: (1) IFNAR, STAT1, and STAT2 mRNA expression levels were slightly higher in the IFN alpha1b group than those in the IFN alpha2b group (P > 0.05). The mRNA expression levels in IFN alpha1b or IFN alpha2b groups were significantly higher than in the IFN alpha2a group (P < 0.05). (2) The PKR mRNA expression showed no significant differences among IFN alpha1b, IFN alpha2b, and IFN alpha2a groups. (3) The RNase L mRNA expression was very weak. We could not compare the differences of the RNase L mRNA levels in different groups by RT-PCR. Western blot results: (1) The IFNAR, and STAT1 protein expressions were greatly up-regulated after IFN alpha induction compared with the untreated group (P < 0.05). (2) The IFNAR, and STAT1 protein expression levels in IFN alpha1b group were slightly higher than the IFN alpha2b group. IFNAR, and STAT1 protein levels of IFN alpha1b or IFN alpha2b group were significantly higher than IFN alpha2a group (P < 0.05). CONCLUSION: STAT1, STAT2, IFNAR mRNA and protein expressions could all be markedly up-regulated after IFN alpha treatment. Effects of IFN alpha1b or IFN alpha2b were greatly stronger than IFN alpha2a. The PKR mRNA expression also was greatly up-regulated after IFN alpha treatment. Expression levels of PKR in IFN alpha1b, IFN alpha2b, and IFN alpha2a groups were all similar. The mRNA level results were consistent with the protein level results. Our results showed that the antiviral activity of IFN alpha1b or IFN alpha2b were stronger than that of IFN alpha2a. The signal transduction molecules STAT1, STAT2, and IFNAR could be regarded as a key index to evaluate antiviral activity of IFN alpha. Further confirmation is still needed to see whether PKR could be regarded as a key index.