Investigation On Pulsing Dendritic Cells With Hepatitis B Virus Vaccine And Antigens Of Elemene Combo-tumor Cell Vaccine Of Hepatic Cell Carcinoma

Objective:Hepatocellular carcinoma (HCC) is one of most common malignant tumors. The relationship between persistent infection of hepatitis B virus (HBV) and pathogenesis of HCC has been well documented, HCC can be considered as an end-stage outcome of HBV infection. Dendritic cells are regarded as the most effective antigen presenting cells. Clinically application of dendritic cells-based tumor vaccine was performed in HCC. Current studies on the DC vaccine of HCC aim to improve preparation, especially, the approach to pulse DC. In the experiment DCs from human peripheral blood mononuclear cells (PBMC) were pulsed with HBV vaccine and or antigens of elemene combo tumor cell vaccine (EC-TCV), their antitumor activity was measured.Methods and Results:1.Culture of DCs:Cell suspension rich in CD14+ were made with adherent method from PBMC and cultured with recombinant human GM-CSF(20ng/ml) and IL-4 (20ng/ml) at 37℃,5% CO2 for 6 days. Half adherent cells were collected. The positive percent of CD83 was 43.84%, the positive percent of HLA-DR was 76.46%. The result showed that the suspension was rich in DC cells.2.Pulsing DC with antigens:Human hepatocarcinoma cell SMMC-7721 cells were treated with elemene and mitomycin C to prepare the EC-TCV. Cell lysates of EC-TCV was made with repeatedly freezing and thawing cells. GM-CSF and IL-4 induced DCs were pulsed with HBV vaccine, cell lysates of EC-TCV or both. Flowcytometry assay showed that CD83 positive percent was 57%,61.01% and 80.69%,no significant changes of HLA-DR expression was observed.3.Allogeneic mixed lymphocytes reaction (MLR):DCs were used as stimulator cells,normal allogeneic nonadherent lymphocytes were used as reactioner cells. Two kind cells were mixed and cultured for 72 hours at 37℃,5% CO2.Cell proliferation was measured with MTT assay. The results showed that DCs pulsed with HBV vaccine, cell lysates of EC-TCV or both gained more potent stimulating capacity to allogeneic lymphocytes, stimulating index was 32.89±4.07%,42.76±3.24%和51.71±2.76, the difference in OD570 among groups was significant (P<0.05)。4.Cytotoxicity of allogeneic lymphocytes stimulated by DC:allogeneic lymphocytes of same donors were stimulated by DCs pulsed with HBV vaccine, cell lysates of EC-TCV or both and were used as effector cells, SMMC-7721 cells were used as targator cells. Two kinds of cells were mixed at 10:1 ratio and cultured for 72 hours at 37℃,5% CO2.Cytotoxicity was measured with MTT assay. The cytotoxcicity of mixed cell suspension was 32.89±4.07%,42.76±3.24% and 51.71±2.76, if DCs were not pulsed that was 20.04±4.24%, the difference among groups was significant (P<0.05)。Results:DCs pulsed with HBV vaccine, cell lysates of EC-TCV or both exhibited the morphological feature and capacity to stimulate allogeneic lymphocytes proliferation and cytotoxicity against SMMC-7721. The protocols used in the experiment may be used treatment of HCC.