Killing Effect Of Dendritic Cells Transfected With Human Aspartat-Î'-hydroxylase Gene On Hepatocellular Carcinoma Cell SMMC-7721 In Vitro

Objective : To investigate the killing effect of cytotoxic T lymphocytes(CTLs) on hepatocellular carcinoma cells line SMMC-7721 after induced by immature dendritic cells(imDCs) transfected with human aspartate-β-hydroxylase gene recombinant adenovirus in vitro.Methods:Immature dendritic cells, from the bone marrow of the mice C57BL/6, loaded with human aspartate β-hydroxylase recombinant adenovirus were used to prepare for DCs vaccine. They were classified as immature dendritic cells infected by Ad-ASPH-IRES2-EGFP(DCs-ASPH group),immature dendritic cells infected by Ad-IRES2-EGFP(DCs-GFP group) and immature dendritic cells group(imDCs group).The expressions of ASPH protein and mRNA in each DCs were detected by Western blot and real-time PCR respectively, and the phenotypic changes of mature dendritic cells were examined by flow cytometry(FCM). The spleen lymphocytes were separated from the same mice C57BL/6,and the surface expressions of specific molecules were detected by flow cytometry(FCM) in the spleen lymphocytes.The specific CTLs induced by DCs vaccine were subsequently co-cultured with hepatocellular carcinoma cells line SMMC-7721. The killing effect of cytotoxic T lymphocytes(CTLs) on SMMC-7721 cells was detected by cell counting kit 8(CCK-8)and flow cytometry. The level of IFN-γ was measured by enzyme-linked immunosorbent assay(ELISA).Results : DCs was successfully cultured and DCs vaccine was constructed.The ASPH protein and mRNA had high expression in DCs-ASPH group.The DCs-ASPH group expressed significantly higher level of CD80,MHCⅡand CD11 c than those in DCs-GFP group and imDCs group. The surface expression of CD3, CD4 and CD8 in the spleen lymphocytes were respectively 79.24%,43.23%,28.93%.The proliferation of SMMC-7721 cells co-cultured with CTLs induced by DCs-ASPH group was lower while the apoptosis rate was higher than other groups.The secretion level of IFN-γ in the CTLs induced by DCs-ASPH group was also significantly higher than those in other groups,and the difference was statistically significant(P?0.05).Conclusion : CTLs induced by DCs transfected with Ad-ASPH-IRES2-EGFP had generated a apparently killing effect on SMMC-7721 cells in vitro.