The Anti-hcc Effect Of CTL Induced By DC Sensitized By SMP30 Combined With HSP70L1 In Vitro

Objective:To prepare a DC loaded with human liver cancer-associated antigen SMP30-HSP70L1,And to explore the in vitro anti-hepatocarcinogenic effects of CTL induced by DC.Methods:(1)SMP30 and HSP70L1 genes were ligated into lentiviral vectors by gene recombination technology to construct SMP30,HSP70L1 fusion(SMP30-HSP70L1)recombinant lentivirus,SMP30 recombinant lentivirus,HSP70L1 recombinant lentivirus.Reexamination of lentivirus titers by fluorescence dilution method.(2)Mononuclear cells were isolated from the peripheral blood of healthy adults with HLA-A2 positive by Ficoll gradient density centrifugation,and DC were induced by cytokines GM-CSF and IL-4 and identified by flow cytometry.(3)DC were transfected by SMP30-HSP70L1 recombinant lentivirus,SMP30 recombinant lentivirus,HSP70L1 recombinant lentivirus and empty lentivirus,liver cancer tissue total proteins(SMP30,HSP70L1 are expressed)were sensitized to DC,and a blank control group was established.The experiment was divided into 6 groups:SMP30-HSP70L1 group,SMP30 group,HSP70L1 group,empty lentivirus group,liver cancer proteins group and blank control group.(4)The expression of SMP30 protein and HSP70L1 protein in DC was detected by Western blot,the secretion of IL-12 and IFN-y in DC was detected by ELISA,and the expression of immune molecules CD80 and CD86 on DC surface was detected by flow cytometry.(5)Autologous T lymphocytes were isolated by human CD3+ T cell magnetic bead sorting kit,and then co-cultured with DC of the above groups to induce CTL,ELISA was used to detect the secretion of cytokines IFN,TNF in CTL,and CCK8 was used to detect the proliferation of CTL in each group.(6)CTL of the above groups were co-cultured with human hepatoma cell line SMMC7721,and flow cytometry was used to detect the anti-hepatoma effect of CTL in vitro.Results:(1)The SMP30,HSP70L1 fusion(SMP30-HSP70L1)recombinant lentivirus,SMP30 recombinant lentivirus and HSP70L1 recombinant lentivirus were successfully constructed.The results of lentivirus titer reexamination showed that the virus titer was about 3×108 TU/ml.(2)Human peripheral blood DC was successfully induced and cultured.After transfecting DC with recombinant lentivirus,the results of western blot showed that the expression levels of SMP30 protein in DC of SMP30-HSP70L1 group and SMP30 group were significantly higher than that of other groups of DC;and the expression levels of HSP70L1 protein in DC of SMP30-HSP70L1 group and HSP70L1 group were higher than other groups(P<0.05).The results of flow cytometry showed that the expression of CD86 in SMP30-HSP70L1 group was higher than that in HSP70L1 group,empty lentivirus group and blank control group(P<0.05),and there was no statistical difference between the SMP30-HSP70L1 group and the liver tissue total protein group.There was no statistical difference in the expression of surface immune molecule CD80 among the groups of DC.The results of ELISA showed that the amount of cytokine IL-1 in SMP30-HSP70L1 group was higher than that in SMP30 group,HSP70L1 group,empty lentivirus group and blank control group(P<0.05),and there was no significant difference in DC between SMP30-HSP70L1 group and total protein group of liver cancer tissues.The secretion of DC cytokines IL-12 and IFN-yin SMP30-HSP70L1 group was higher than that in HSP70L1 group,empty lentivirus group and blank control group(P<0.05),and there was no statistical difference between SMP30-HSP70L1 group and liver cancer tissue total protein group and SMP30 group.(3)The purity of T lymphocytes isolated by immunomagnetic beads was 93.1%.The TNF-aand IL-12 secreted by CTL induced by DC in SMP30-HSP70L1 group were higher than other groups(P<0.05),except for the total protein group of liver cancer tissues.The IFN-a and IFN-y secreted by CTL induced by DC in SMP30-HSP70L1 group were higher than those in blank control group and empty lentivirus group(P<0.05),and there was no significant difference between SMP30-HSP70L1 group and the other three groups.The results of CCK8 cell proliferation assay showed that the SMP30-HSP70L1 group was higher than the blank control group,the empty lentivirus group,the 70 lentivirus group and the 30 lentivirus group(P<0.05),and there was no significant difference between SMP30-HSP70L1 group and liver cancer tissue protein extract group.(4)The results of flow cytometry showed that:when the target ratio(E/T)was 15:1,the killing rate of CTL induced by DC in SMP30-HSP70L1 group on SMMC7721 cells(14.50±0.33)%was higher than that of the SMP30 group,the HSP70L1 group,the empty lentivirus group and the blank control group(P<0.05),and there was no statistical difference between the SMP30-HSP70L1 group and the total protein group of cancer tissues(14.25±0.64)%.when.the target ratio(E/T)was 30:1,the killing rate of CTL induced by DC in SMP30-HSP70L1 group on SMMC7721 cells(14.50±0.33)%was higher than that of the HSP70L1 group,the empty lentivirus group and the blank control group(P<0.05),and there was no statistical difference between the SMP30-HSP70L1 group and the total protein group(50.38±0.48)%,SMP30 group(46.42±2.14)%.Conclusion:Compared with other experimental groups except the total protein group of liver cancer tissue,DCs co-modified by human liver cancer-associated antigen SMP30 and HSP70L1 can promote the maturation of DC and stimulate the proliferation and activation of CTL more effectively.And the CTL induced by DC in SMP30-HSP70L1 group can kill hepatoma cell line SMMC7721 better in vitro when the E/T is 15:1.