Construction Of Interferon-resistant HBV Cell Model

Objective: To establish an interferon-resistant hepatitis B virus cell model and provide experimental basis for further investigating the mechanism of HBV resistance to interferon.Methods: 1.HepG2.2.15 was continuously grown in the presence of 10,30,50 and 70 IU/m L of IFN?-2b for up to 48 weeks,the HepG2.2.15/IFN?-2b cell model was constructed after 48 weeks of induction.2.HepG2.2.15 cells were treated with different concentrations of IFN?-2b(250,500,750,1000,1250 and 1500 IU/m L,respectively),The expression of HBs Ag,HBe Ag,HBV DNA in the supernatant of cell culture medium were compared after treated with different concentrations of IFN?-2b to ensure the best effective concentration.3.The cells were treated with the best-effect concentration.The expression of HBs Ag,HBe Ag in the supernatant of cell culture medium before and after the treatment were compared by ELISA,the absolute quantity of HBV DNA in the supernatant of cell culture medium before and after the treatment were determined by quantitative realtime PCR assay.4.MTT assay was used to compare the inhibitory effects of IFN?-2b on HepG2.2.15 and HepG2.2.15/IFN?-2b cells proliferation.5.The relative expressions of OAS,Mx A and ISGF3 m RNA of the HepG2.2.15 and HepG2.2.15/IFN?-2b cells which treated with the best-effect concentration were evaluated by RT-PCR.Results: 1.After the HepG2.2.15 cells were treated with IFN?-2b,the secretions of HBs Ag,HBe Ag,and HBV DNA were all inhibited to different degrees in a dose-dependentmanner.The inhibition rate on HBs Ag,HBe Ag and HBV DNA replication peaked at the concentration of 1250 IU/m L(the inhibition rate was 72.2%,49.3% and 58.8%,respectively.).When the concentration of IFN?-2b was 1500 IU/m L,the inhibition rate on HBs Ag,HBe Ag and HBV DNA increased slowly.Therefore,the findings suggested that the best-effect concentration of IFN?-2b was 1250 IU/m L.2.After stimulation with low concentrations of IFN?-2b for 12 weeks,the 50 IU/m L group showed significant resistance to the best-effect concentration of IFN?-2b.Compared with the levels before stimulation,the inhibition rate on HBs Ag,HBe Ag and HBV DNA decreased by 25.48%,8.40% and 15.43%,respectively,suggesting that 50 IU/m L was the best-stimulation concentration.3.After stimulated with 50 IU/m L IFN?-2b for 12~48 weeks,the results showed that the inhibition rate on HBs Ag,HBe Ag and HBV DNA after 36 weeks was the most significant.4.The growth inhibition of HepG2.2.15 and HepG2.2.15/IFN?-2b cells were observed after treatment of IFN?-2b and the inhibition rate were dose-dependent(p<0.05),with the same dose of IFN?-2b,the inhibition rate were equivalent(p>0.05),Its IC50 was 262284.63 and 226471.20 IU/m L,respectively.5.After treated with 1250 IU/m L IFN?-2b,compared with HepG2.2.15,the relative expressions of OAS,Mx A and ISGF3 m RNA of the HepG2.2.15/IFN?-2b have different degree of reduced.Conclusion: Continuous induction with 50 IU/m L IFN?-2b for 36 weeks could most easily induce drug resistance in HepG2.2.15 cells.