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Published on: March 22, 2011
[Recombinant adeno-associated virus carrying hepatitis B virus C gene (rAAV-HBV-C) effectively pulses dendritic cells
Min Cong1, Ping Wang, Zhong-Yu Yan
1Liver Disease Research Center, Beijing Friendship Hospital, Capital University of Medical Sciences, Beijing 100050, China.
Objective:
Recombinant virus pulsated dendritic cells (DCs) may affect their survival, growth and maturity. This study is to test the infection efficiency of recombinant adeno-associated virus carrying hepatitis B core antigen (rAAV-HBV-c) to DCs and the growth and maturity of them.
Methods:
Peripheral blood mononuclear cells (PBMCs) were isolated from healthy blood donors. Adherent monocytes were pulsed by rAAV-HBV-c and 293 lysate as controls on the first day of isolation. DCs were cultivated in AIM-V media with 1000 u/ml granulocyte macrophage stimulating factor (GM-CSF), 1000 u/ml interleukin-4 (IL-4) and 50 ng/ml tumor necrosis factor-alpha (TNFalpha) separately in vitro. DCs were examined at different times and the expressions of several clusters of differentiations (HLADR, CD14, CD80, CD83, CD86) were studied using FACS after being cultured for 7 days. The transcription and expression of HBV-C gene were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and intracellular staining fluorescence activated cell sorter (FACS), respectively.
Results:
The rAAV-HBV-c infected and uninfected monocytes gradually matured and their morphology had no significant differences. The CDs expressed on the surfaces of the two groups of DCs were also similar (HLADR: 96.1% vs. 94.5%; CD86: 87.7% vs. 89.8%; CD83: 75.6% vs. 78%; CD80: 52% vs. 54.3%; CD14: 6.4% vs. 4.5%). HBV-C gene mRNA expression was measured using RT-PCR and 89.5% of the rAAV-HBV-c infected DCs showed their protein expression using FACS.
Conclusion:
rAAV-HBV-c can effectively pulse DCs without affecting the growth and maturity of them.
