The Study Of Immunological Properties Of The Human Dendritic Cells After Loaded With HBV Antigen In Vitro

Despite the availability of efficient vaccines for protecting previously unexposed individuals, hepatitis B virus infection remains a major cause of morbidity and mortality worldwide. Two therapeutic agents are currently recommended for the treatment of chronic hepatitis B virus, Interferon-alfa (TFN- a ) and Lamivudine. But neither IFN- a nor Lamivudine can clear covalently closed circular DNA (cccDNA) of HBV. After therapy was stopped, HBV DNA reappeared in most patients. It is worth noticing that these therapy depend on the lever of patients' own immunology function too much, and their mechanisms of inhibiting viral replication or stimulating cell mediation immunity to HBV have not been fully delineated.The accumulated data show that cytotoxic T lymphocytes (CTLs) activity play an important role in resolving HBV infection. Dendritic cells are the most potent antigen presenting cell in vivo. DCs play a central role in both humoral and cellular immunity. The study of the DC based immunotherapy provides us a promising way to cure the chronic HBV infection.Firstly, Monocytes were isolated from normal and CHB peripheralblood mononuclear cells (PBMCs), cultured with cytokines including GM-CSF, IL-4,TNF- a and IFN- a to induce and expand mature DCs. Secondly, we put the proper dote of HBsAg or HBcAg into the culture. Thirdly, the mature DCs were loaded with HBV antigen. Then we detected the intracellular cytokines of the DC induced T cell through four-colour flow cytometry. Results are showed briefly as below:1. The monocyte derived DC cultured in GM-CSF, IL-4, TNF-a, and IFN- a havest in seven days later. Surface marker assay by FACS showed that DCs expressed CDla (55.53%), CD80(83.78%), DR (98.76%), CD86( 99.08%), CD83 (64.70%) and CD40(76.23%). Mixed lymphocyte reaction (MLR) suggested that DCs induced through such cytokines cocktail were strongly potent to stimulate the proliferation of T lymphocytes.2. Characterize the antigen loaded DCs by immunohistochemistry. Surface marker assay by FCM showed that there was no difference between the antigen loaded DCs and none antigen loaded DCs. DCs pulsed with antigen and controls were cultured with autologous lymphocytes at the radio of 1:10 for 5 days. Then we observed the production of intracellular cytokine, IFN- Y and IL-4 in the DCs induced T cells by FCM detection.Our results demonstrated that we load the DCs with the HBVantigens successfully. The DCs pulsed with HBsAg or HBcAg were matured and could induce the Thl and Tel profile both health and CHB.And the DCs of healthy donor which HBsAb positive pulsed with HBsAg could even induce a higher level Thl and Tcl type cytokine than them pulsed with HBcAg. However, the cultured DCs of CHB have a low lever of immunophenotype markers on their surface.And the lever of Antigen pulsing with the DCs of CHB also resulted in a decreased capacity to present antigen to autologous T cells and to induce Thl and Tcl.