Antitumor Effect Of Immunization With Fusion Of Dendritic Cells And AFP~+GP73~+HepG2 Against Hepatocellular Carcinoma

Objective:Surgery,radiotherapy,chemotherapyandbiological immunotherapy are currently the main means of treating malignant tumors.Among these therapeutic approaches,immunotherapy based on dendritic cells(DCs)has became the focus.DC/tumor cell hybrid vaccine has been extensively studied for tumor therapy.However,the low immunogenicity of tumor cells and the low expression of antigen are the key factors affecting the anti-tumor effectiveness.Alpha Fetoprotein(AFP)is the most widely used markers of Hepatocellular carcinoma(HCC)and is used as a target antigen for HCC immunotherapy.DC loaded with AFP can induce AFP-positive HCC cytotoxic T lymphocytes(CTL)response.Golgi protein 73(GP73)is the most valuable HCC marker found in recent years.The preliminary results of our study showed that the level of serum GP73 in HCC patients was significantly higher,and the sensitivity and specificity of GP73 in diagnosing HCC was significantly higher than that of AFP;And the results of immunohistochemistry suggest that liver cancer can produce a large number of GP73,suggesting that it can be used as molecular markers of HCC.To further clarify whether GP73 can be used as a target antigen for HCC immunotherapy,improve the immunogenicity of hepatocellular carcinoma cells combined with AFP and enhance the anti-tumor immune response of DC/tumor cell fusion vaccine,we used gene engineering to construct hepatocarcinoma cells which is overexpressed AFP and GP73,and then used polyethylene glycol(PEG)to fuse DC and constructed hepatocarcinoma cells to observe the anti-tumor effects of DC/tumor cell hybrid vaccine in the treatment of hepatocellular carcinoma.Methods:The lentivirus carrying AFP and GP73 gene was constructed by gene engineering technique,and the expression of AFP and GP73 of Hepatocellular carcinoma cell lines were detected by flow cytometry.The optimal conditions of virus infection of hepatocarcinoma cells were determined by pre-experiment of virus infection.Under the optimal conditions,the two kinds of lentiviruses were transfected into hepatocarcinoma cells respectively or co-transfected,and the transfection efficiency was observed by inverted fluorescencemicroscope.TheexpressionofAFPandGP73in AFP~-GP73~-Hep G2,AFP~+GP73~-Hep G2,AFP~-GP73~+HepG2and AFP~+GP73~+HepG2 cells were detected by real-time PCR(RT-PCR)and Western blot.The peripheral blood mononuclear cells of healthy volunteers were isolated by density gradient centrifugation,and were induced by cytokines to DC.AFP~+GP73~+HepG2 cells and DCwere induced by PEG to generate AFP~+GP73~+HepG2/DC cell hybrid vaccine.Flow cytometry was used to analyze the expression of a number of surface markers on AFP~+GP73~+HepG2/DC cell hybrid vaccine.The ability of CTL cells to kill target cells in vitro was measured by CCK-8 kit.The in vivo anti-tumor effects of AFP~+GP73~+HepG2/DC cell hybrid vaccine were investigated in the HCC model in mice.Results:AFP~-GP73~-HepG2 hepatoma cells were screened and were transfected with lentivirus.The transfection efficiency was above 85%by fluorescence inversion microscope.RT-PCR and Western blot showed that AFP was highly expressed in AFP~+GP73~-Hep G2 and AFP~+GP73~+HepG2 cells,and GP73 was highly expressed in AFP~-GP73~+HepG2 and AFP~+GP73~+HepG2 cells,suggesting successful transfection.Peripheral blood mononuclear cells under the action of cytokines can be induced to differentiate into cells with typical dendritic morphology.The fusion cells,AFP~+GP73~+Hep G2/DC,exhibited high levers of expression of CD80,CD86,and HLA-DR.The CTL induced by AFP~+GP73~+HepG2/DC has a significant cytotoxic effect on hepatoma cells in vitro and subcutaneous injection of AFP~+GP73~+HepG2/DC in HCC mice efficiently inhibited the tumor growth.Conclusion:GP73 can be used as a target antigen for HCC immunotherapy and can improve the immunogenicity of hepatocellular carcinoma cells combined with AFP and enhance the anti-tumor immune response of DC/tumor cell hybrid vaccine.Therefore,this study provides a new strategy for the treatment of AFP-negative hepatocellular carcinoma.