| Hepatitis B virus infection is a worldwide epidemic, and it is a serious public health problem. Although the pathogenesis of hepatitis B is not completely clear, but due to HBV and not as a direct result of the pathological changes of hepatic cells, researchers generally believe that body of pathological injury and disease outcome is related to virus invasion triggered an immune response, for HBV antigens in immune response strength determines the final disease outcome. Therefore, the detection of specific CD4+/CD8+T cell responses will help to assess the pathological and clinical outcome of HBV infection.This research will use the autophagy inducing agent Huh7.6 cells, HepG2.2.15 cell, raise containing multiple HBV antigen component of the autophagic bodies HBV+DRibbles and the detection of HBV+DRibbles stimulated PBMC of hepatitis B patient release IFN-y levels, to explore the correlation with the clinical staging and prognosis of the diseaseObjectives:In order to stimulate the PBMC of hepatitis B patients (HBV+DRibbles) in vitro with HBV containing multiple antigens, to investigate:1) whether HBV+DRibbles can stimulate the PBMC and produce cytokines in patients with hepatitis B virus (HBV)? 2) HBV+DRibbles stimulate PBMC of hepatitis B patient to produce cellular factor and how to relate with the clinical stage of hepatitis B patient and disease prognosis?Methods:1. Preparation of human hepatoma cell line Huh7.6 cells, transfected with HBV full gene of human hepatoma cell line HepG2.2.15 cell release of autophagy(DRibbles).2. Transmission electron microscope.was used to detect the ultrastructure of Dribbles Western. Blot used to detect the expression of LC3Ⅰ/Ⅱ in Dribbles.. ELISA and chemiluminescence detection of HBsAg and HBeAg in autophagy bodies3. ELISA detection of HBV+DRibbles to stimulate the different clinical stages of hepatitis B patients with PBMC to produce IFN-γ level4. Statistical analysis of HBV+DRibbles stimulation of hepatitis B patients with IFN-y production of PBMC gamma levels and clinical stage and disease outcome related.Results:1:Human hepatoma cell line, HepG2.2.15 cells, Huh7.6 cells, can release autophagosomes in vitro culture1) Blot Western HepG2.2.15 detection showed that DRibbles cells in the LC3 to LC3II based.2) The presence of a large number of bilayer membrane structures in the extract was found by transmission electron microscopy.3) DRibbles can be raised to HBV antigen composition. HBV antigen content in HBV+DRibbles induced by HepG2.2.15 cells was higher2:HBV+DRibbles can effectively stimulate the hepatitis B patients PBMC IFN-y, and dose-dependent stimulation of HBV+DRibbles in patients with hepatitis B PBMC IFN-y levels were significantly higher than that of HBV DRibbles.3:Acute hepatitis B patients cured HBV+DRibbles stimulated IFN-y levels were significantly higher in patients with chronic hepatitis B, and have a certain relationship with the clinical stage.Conclusions:1. HBV+DRibbles cells were successfully prepared from human hepatocellular carcinoma cell lines (HepG2.2.15) with HBV antigen.2. HBV+DRibbles can effectively stimulate the hepatitis B patients PBMC produce IFN-γ.There is a certain correlation in the IFN-γ level and clinical stage. |
