il 29和ifn α体外抗乙肝病毒作用与机制探讨-study on the anti-hepatitis b virus effect and mechanism of il 29 and ifn α in vitro.docx

安徽医科大学硕士学位论文 安徽医科大学硕士学位论文 PAGE PAGE 10 Effect and Mechanism of IL-29 and IFN-α Against Hepatitis B Virus in vitro Abstract Objective To explore the antiviral effect and mechanism of IL-29 and IFN-α against hepatitis B virus in HepG2.2.15 Method HepG2.2.15 cells were cultured and treated with IL-29 and IFN-α. PCR and Real-time PCR were used to study the antiviral effect of IL-29 against hepatitis B virus by detecting the amount of HBV mRNA expressed and the quantity of mRNA transcribed from genes of Max, 2’5’-OAS and PKR. We analyzed signal pathways induced by IL-29 and IFN-α by Western blot, then used PD98059 and LY294002, as potent and specific inhibitor, to selectively inhibit ERK and AKT phosphorylation respectively. Through these methods we discussed the relationship between the expression of MxA, 2’,5’-OAS, PKR genes and signal pathways. Results In HepG2.2.15 cells, IL-29 and IFN-α could reduce the amount of HBV mRNA, up-regulate MxA as well as 2,5-OAS, and induce the phosphorylation of ERK and AKT.( IFN-α can also increase the expression of PKR). Phosphorylation of ERK and AKT were clearly respectively blocked by PD98059 (100 μM) and LY294002 (50 μM), even when the cells were pretreated with these inhibitors for one hour prior to stimulated by IL-29 and IFN-α. When the phosphorylation of AKT was blocked by LY294002, IFN-induced mRNA level expression of the MxA, 2’,5’-OAS and PKR decreased. The block of phosphorylation of ERK1/2 by PD98059 alone resulted in about 3 times increase of the MxA（which was induced by IL-29 or IFN-α）, but it had no significant effect on 2’,5’-OAS as well as PKR. Conclusion These findings show that IL-29 and IFN-α have obvious antiviral activity towards HBV in HepG2.2.15 cells. In research of the mechanism, we have found that the mRNA level expression of MxA, 2’,5’-OAS and PKR is up-regulated by PI3K-AKT signal pathway. And Raf-MEK-ERK signal pathway has negative regulatory effect on the mRNA level expression of MxA in HepG2.2.15 ce