| Objective:To construct a lentiviral recombinant that mediates SMP30 gene of mouse liver cancer-related antigen,and to investigate the efficacy of SMP30in sensitizing DC at gene level and its effect in inducing CTL to resist HCC in vitro.Methods:(1)SMP30 gene was ligated into lentiviral expression vector LV5with green fluorescent protein(GFP)by gene recombination technique.The recombinant was constructed and transfected into mouse DC.The experiment was divided into four groups:empty lentiviral group(LV-control group),SMP30lentiviral group(LV-SMP30group),SMP30 protein group(SMP30protein group)and blank control DC group(DC group).The expression of SMP30 in transfected group was detected by western blot.(2)Flow cytometry was used to detect the differences of surface molecules of sensitized DC among groups,andtodetermine the influence of SMP30 lentivirus recombinants on DC maturation.(3)The levels of IL-12 and IFN-?in DC supernatant were detected by ELISA.(4)The CD8~+T cell magnetic bead sorting kit sorts CD8~+T cells from the single cell suspension of BALB/c mouse spleen,and then the purity of CD8~+T cells was detected by flow cytometry,and then co-incubated with DC cells of the above groups to induce(CTL),and the secretion of IL-2 and IFN-?in the supernatant of CTL of each group was detected by ELISA.(5)The induced CTL was incubated with H22 hepatocarcinoma cells of mice,and the killing effect of CTL on hepatocarcinoma cells was detected by flow cytometry.Results:(1)SMP-30 lentiviral recombinant was successfully constructed and transfected into mouse DC.Western blot showed that the expression of SMP30 protein located in LV-SMP30 group(2)The expression of CD80 and CD86on DC surface in LV-SMP 30 group was significantly higher than that in DC group and LV-control group(P<0.05).The expression of CD80 and CD86 on DC surface in SMP 30 protein group was significantly higher than in DC group and LV-control group(P<0.05).There was no significant difference between DC group and LV-control group.There was no significant difference between LV-SMP30 group and SMP30 group.(3)After initial sensitization of each group DC 72h,the results of ELISA detection of DC supernatant showed that the levels of IL-12 and IFN-?secreted by DC in LV-SMP30 group were significantly increased,and the difference was statistically significant compared with DC group and LV-control group(P<0.05).The levels of IL-12 and IFN-?secreted by DC in SMP 30 protein group were significantly higher than those in DC group and LV-control group(P<0.05).There was no significant difference between DC group and LV-control group.There was no significant difference between LV-SMP30 group and SMP 30 protein group.However,after one week of continuous culture of DC in each group,the levels of IL-12 and IFN-?secreted by DC in LV-SMP 30 group were significantly higher than those in SMP 30 protein group by ELISA,and the difference was statistically significant(P<0.05).(4)The results of flow cytometry showed that CD8~+T cells were obtained by isolating the single cell suspension of BALB/c mouse spleen,accounting for 98.55%of the total T cells.(5)72 hours after CTL was induced by DC in each group with initial sensitization,the results of detecting CTL supernatant by ELISA showed that the levels of IL-2 and IFN-?secreted by CTL in LV-SMP30 group increased significantly,and the difference was statistically significant compared with DC group and LV-control group(P<0.05).The levels of IL-2 and IFN-?secreted by CTL in SMP 30 protein group were also higher than those in DC group and LV-control group.There was no significant difference between DC group and LV-control group.There was no significant difference between LV-SMP30 group and SMP 30 protein group.But after one week of continuous induction of CTL in each group,the levels of IL-2 and IFN-?secretion in CTL of LV-SMP30 group were significantly higher than those of SMP 30 protein group,and the difference was statistically significant(P<0.05).(6)Flow cytometry showed that DC-induced CTL cells of LV-SMP30 group with initial sensitization of DC at the fifth day had significant killing effect on H22 mouse hepatoma cells,and the killing rate was higher than that of SMP30 protein group,DC group and LV-control group,the difference was statistically significant(P<0.05).The killing rate of SMP30 protein group was significantly higher than that of DC group and LV-DC group(P<0.05).There was no significant difference between DC group and LV-DC group.Conclusion:SMP30,as a liver cancer-related antigen,induces CTL by sensitizing DC through lentiviral mediator,and can produce better killing effect on H22 mouse liver cancer cells compared with CTL induced by DC directly sensitized by SMP 30 protein. |
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