title: [Establishment of a cell co-culture system in accordance with the immunological characteristics of chronic HBV infection].
abstract: Objective:To investigate whether the co-culture of Huh7.93 cells and peripheral blood mononucleated cells from chronic hepatitis B patients (cPBMCs) can simulate the replication features of hepatitis B virus (HBV) and immune function in chronic hepatitis B (CHB) patients, and to provide an in vitro cell co-culture system for the research on immune clearance in chronic HBV infection.Methods:Huh7.93 cells were cultured alone or co-cultured with peripheral blood mononucleated cells from healthy people who underwent physical examination (nPBMCs) or cPBMCs for 7 days. The CCK8 assay was performed to measure the proliferative activity of Huh7.93 cells, and quantitative real-time PCR and Southern blot were used to measure HBV replication in cPBMCs and co-cultured cells. The independent samples t-test was used for comparison between two groups.Results:When Huh7.93 cells were co-cultured with peripheral blood mononucleated cells (PBMCs) at a certain ratio, Huh7.93 cells had a high proliferative activity and good cell growth. HBV could not infect or replicate in cPBMCs. HBV DNA in the supernatant of Huh7.93 cells co-cultured with cPBMCs showed significant increases and significantly higher than that in the supernatant of Huh7.93 cells cultured alone on day 4 (6.01 ± 0.20 log10copies/ml vs 4.99 ± 0.08 log10copies/ml,P= 0.000) and day 7 (7.82 ± 0.24 log10copies/ml vs 6.96±0.09 log10copies/ml,P= 0.000). On day 7 of culture, the cell lysis buffer of Huh7.93 cells co-cultured with cPBMCs had a significant increase in the level of HBV replicative intermediate compared with that of Huh7.93 cells cultured alone. After HepG2.2.15 cells were co-cultured with cPBMCs, there was no significant increase in HBV replication.Conclusion:The co-culture of Huh7.93 cells and cPBMCs produces similar viral replication as human body infected with HBV and can well simulate the liver microenvironment and immune function in CHB patients, which provides a new method for the research on immune clearance in chronic HBV infection.
authors.affiliation: The Liver Disease Diagnosis and Treatment Center of PLA, Bethune International Peace Hospital, Shijiazhuang 050082, China.
authors.forename: H J
authors.initials: HJ
authors.lastname: Li
authors.forename: X Y
authors.initials: XY
authors.lastname: Yang
authors.forename: F B
authors.initials: FB
authors.lastname: Kang
authors.forename: F X
authors.initials: FX
authors.lastname: Liu
authors.forename: D X
authors.initials: DX
authors.lastname: Sun
mesh_terms: D018920:Coculture Techniques; D004261:DNA Replication; D004279:DNA, Viral; D006515:Hepatitis B virus; D019694:Hepatitis B, Chronic / Q000276:immunology*; D006801:Humans; D036102:Peripheral Blood Stem Cell Transplantation; D060888:Real-Time Polymerase Chain Reaction; D014779:Virus Replication
chemical_list: D004279:DNA, Viral