Human Dendritic Cells Were Modified By SMP30-HSP70L1 To Induced Cytotoxic T Lymphocytes Against Hepatocellular Carcinoma In Vivo

Objective:To investigate the anti-tumor effect of cytotoxic T lymphocyte(CTL)induced by dendritic cell(DC)loaded with human HCC associated antigens senescence marker protein-30(SMP30)and heat shock proteins 70 like1(HSP70L1)on experimental hepatocellular carcinoma in vivo.Methods:(1)Peripheral blood mononuclear cell(PBMC)were isolated from HLA-A2~+healthy adult peripheral blood by Ficoll density gradient centrifugation.DC was induced by recombinant human GM-CSF,IL-4 and TNF-α.SMP30 and HSP70L1 genes were connected to lentivirus vector by gene recombination technology to construct SMP30 and HSP70L1 double genes(SMP30-HSP70L1)recombinant lentivirus,SMP30 recombinant lentivirus and HSP70L1 recombinant lentivirus.DC was sensitized with SMP30-HSP70L1recombinant lentivirus,SMP30 recombinant lentivirus,HSP70L1 recombinant lentivirus,no-load lentivirus and hepatocarcinoma tissue total protein(HCC tissue total protein),respectively.Western blot was used to detect the expression of SMP30 and HSP70L1 in each group.(2)Autogenous T lymphocytes were isolated by CD3 Micro Beads,and CTL was induced by co-culture of CD3~+T lymphocytes and DC.(3)Nude mice transplanted with SMMC-7721 were randomly divided into 7 groups,6 in each group:SMP30-HSP70L1 group,HCC tissue total protein group,SMP30 group,HSP70L1 group,lentivirus empty group,blank DC group and PBS group.(4)The first CTL was infused on 12d after subcutaneous transplantation of tumor-bearing nude mice,and then the CTL was infused every 4 days(i.e.on the 5d,9d,13d and 17d after the first CTL infusion),5 times in total,during which the length and diameter of subcutaneous transplantation tumors were measured with a vernier caliper,the weight of tumor-bearing nude mice was weighed by electronic balance,and the growth curve and weight change curve of transplanted tumors were drawn.(5)Tumor-bearing nude mice were sacrificed on the second day after the last CTL infusion.Tumor inhibitory rates were calculated at the same time.Morphology of Subcutaneous transplanted tumor,liver and kidney were observed by hematoxylin-eosin(HE)staining.(6)The concentrations of IFN-γand TNF-ɑin the serum of tumor-bearing nude mice were detected by ELISA.Results:(1)WB showed that the relative expression levels of SMP30protein in SMP30-HSP70L1 group and SMP30 group were higher than those in HCC tissue total protein group,HSP70L1 group,lentivirus empty group,blank DC group,and the differences were statistically significant(P<0.05).The relative expression levels of HSP70L1 protein in HCC tissue total protein group and SMP30-HSP70L1 group were higher than those in SMP30 group,lentivirus empty group and blank DC group.HSP70L1 group was higher than those in lentivirus empty group and blank DC group,and the differences were statistically significant(P<0.05).(2)At the end of CTL infusion in tumor-bearing nude mice,the growth of subcutaneous tumors were the fastest in PBS group.The volume of subcutaneous transplanted tumors in SMP30-HSP70L1 group was the smallest.The growth of subcutaneous tumors in PBS group increased significantly on 9d,while the growth of subcutaneous tumors in other groups did not increase.On 15d before CTL treatment,there was no significant difference in growth degree of subcutaneous transplanted tumors in all groups except PBS group.After 15d of CTL treatment,the growth degree of transplanted tumors in each group began to show differences.(3)Tumor inhibition rates of SMP30-HSP70L1 group,HCC tissue total protein group and SMP30 group were 58.57%,55.48%and 56.77%,respectively,while HSP70L1 group was 47.10%,but only 28.39%and 26.45%in lentivirus empty group and blank DC group.It showed that CTL induced by DC loaded with SMP30-HSP70L1 had a certain extent anti-tumor effect,but there was no significant difference in the final tumor weight of HCC tissue total protein group,SMP30 group and HSP70L1 group(P>0.05).(4)After HE staining,it can be seen that tumor cells in each group are diffusely distributed,closely arranged,with little stroma and obvious cell heteromorphism.There are more angiogenesis in PBS group,blank DC group and lentivirus empty group,and less blood vessels in SMP30-HSP70L1 group and HCC tissue total protein group.(5)The concentration of IFN-γ(531.22±2.39 pg/m L)of SMP30-HSP70L1 group was higher than that of HCC tissue total protein group,SMP30 group,HSP70L1 group,lentivirus empty group,blank DC group and PBS group(P<0.05).The concentration of TNF-α(121.16±10.07 pg/m L)of SMP30-HSP70L1 group was the highest in each group,which was statistically different from HCC tissue total protein group,lentivirus empty group,blank DC group and PBS group(P<0.05);but compared with SMP30 group and HSP70L1 group,there was no statistical difference(P>0.05).(6)The weight of nude mice in each group did not decrease significantly.HE staining of liver and kidney showed that the cell morphology was relatively complete.The nucleus did not increase,and there was no traumatic pathological reaction.Conclusion:CTL induced by DC was modified with human HCC associated antigen SMP30 and HSP70L1 exert an inhibiting effect on the growth of subcutaneous transplant tumor and change some related anti-tumor cytokines in tumor-bearing nude mice.It has little side effects on the treated nude mice.