Liposome-mediated Hbcag Gene Transfection Transfection Efficiency And Functional Study Of Human Dendritic Cells

BackgroundHepatitis B virus (HBV) is a major human infectious pathogenthat was first discovered in leukemia patients and Australian aboriginalsin 1965. More than one-third of the world's population has been infectedwith HBV. Chronic active hepatitis associated with HBV infection oftenleads to the development of cirrhosis, liver failure, and highly malignantliver cancer. Despite the availability of efficient vaccines for protectingpreviously unexposed individuals, hepatitis B virus infection remains amajor cause of morbidity and mortality worldwide. Two therapeuticagents are currently recommended for the treatment of chronic hepatitis Bvirus, Interferon-alfa (IFN-α) and Lamivudine. But neither IFN-αnorLamivudine can clear covalently closed circular DNA (cccDNA) of HBV.After therapy was stopped, HBV DNA reappeared in most patients. It isworth noticing that these therapy depend on the lever of patients' ownimmunology function too much, and their mechanisms of inhibiting viralreplication or stimulating cell mediation immunity to KBV have not beenfully delineated.Cytotoxic T lymphocytes (CTL) play a key role in the clearance ofviruses and in the control of tumor development. They are firstly able toidentify viral or cancer-associated peptides presented by MHC classⅠmolecules, expressed at the surface of infected or transformed cells, and secondly to kill these cells. Thus, the induction of CTL activity is a majorgoal for vaccine development and immunotherapy.Dendritic cells (DCs) are heterogenous, professionalantigen-presenting cells that are uniquely equipped with molecules andstrategically placed between internal and external environment, whichenable them to link innate and adaptive immunity. DCs are the mostpotent antigen presenting cell in vivo. The study of the DC basedimmunotherapy provides us a promising way to cure the chronic HBVinfection.ObjectiveThe aim of our study is to evaluate the hepatitis B core antigen(HBcAg) expression efficiency in the dentritic cell (DC) derived fromperipheral blood monouclear cells (PBMC) transfected by the cationliposome bearing the hepatitis B core gene, the cytokine IFN-γsecretedby self-lymphocytes what were stimulated by the transfeced DC, and thecapacity to kill HepG2.2.15 cells of the stimulated lymphocytes.Methods:1. PBMC centrifugated by the HES centrifugation were cultured andstimulated into DC by granulocyte-macrophage colony stimulating factor(GM-CSF) and interleukin-4 (IL-4), and their morphology and phenotypewere determinated by the inverted microscope and flow cytometryrespectively. 2. The report gene GFP (rate of DNA to liposome are 1:3, 1:4, 1:5and 1:6) and hepatitis B core gene (rate of DNA to liposome is 1:5) wasrespectively transfected into DC on the fifth day. The expressionefficiency of GFP at 72 hours and expression efficiency of hepatitis Bcore gene at 48, 72, 96 and 120 hours were tested by the flow cytometry.3. Detection of the transfected cell surface markers:collect DC aftertransfected 48 hours,then add monoclonal antibodys as followsHLA-DR-FITC, CD86-PE,CD1a-FITC,CD80-PE,CD83-FITC,CD40-PE. use flow cytometry to detect the cell surface molecules of thetwo groups DC.4. Detection of the HBcAg expression: use immumofluorescencemethod to observe the fluorescent degree of the transfected DC withfluorescence microscope.5. Mixed culture:DCs were divided into three groups as follows:pJW4303/HBc-DC group,pJW4303-DC group and DC group. Mix theself-lymphocyte with above three groups DCs at the ration of 10:1.6. Detection of IFN-γby Elispot: the secretion status of IFN-γinthree groups was measured by Elispot on day 5 after mixed culture.7. CTL (cytotoxicity T lymphocyte)experiment: three groups ofmixed culture cells were used to kill the HepG2.2.15 cell in order tomeasure the CTL capability.Results 1. The morphology of the DC transfected by the liposome did notchange significantly.2. The expression efficiency of the GFP at 72 hours of the fourdifferent DNA to liposome rates mentioned above were 37.12%, 48.55%,52.13% and 50.75% respectively.The expression efficiency of hepatitis Bcore gene at 48, 72, 96 and 120 hours were 31.58%, 55.80%, 54.18% and56.99%.3. The HLA-DR, CD86, CD1a, CD80, CD40, CD83 expressionefficiency of the transfeced DC were 71.6±3.26%,95.86±1.96%,36.43±3.31%,86.70±3.19%,70.13±1.78%,66.70±2.51%, whilecontrol group were 73.15±3.22%,94.83±3.36%,39.35±3.44%,77.73±2.31%,72.73±4.85%,30.63±3.49%. Compared with thenon-transfected DC, CD83 expression efficiency of the transfeced DCincreased (P＜0.05), while else molecules have no difference.(P＞0.05)4. There was flavovirens fluorescence in pJW4303/HBc-DC groupwith fluorescence microscope, while no fluorescence in non-transfectedDC group.5. The lymphocyte after stimulated by pJW4303/HBc-DC couldsecret IFN-γ, while the controls lymphocyte coudn't.6. CTL: The death rates of HepG2.2.15 cells induced by DCstransfeced with pJW4303/HBc were (62.5±4.8)%(E:T=10:1),(71.8±5.3)% (E:T=20:1),(81.5±5.0)% (E:T=40:1), while the pJW4303 transfected DC and non-transfected DC only induced relatively lowercytotoxicity (P＜0.05)ConclusionsThe hepatitis B core gene could be efficiently transfected into DCderived from PBMC by the cation liposome with stable proteinexpression at 72 hours after the transfection. The lymphocyte afterstimulated by pJW4303/HBc-DC could secret IFN-γand coule evoke ahigher CTL response in vitro.