Study On The Kill Activity Of The Whole Tumor Cell Antigen Pulsed DC Co-culture With CIK For Renal Cell Carcinoma Cell

ObjectiveAdopt CCK-8 kit to detect the kill activity of CIK co-culture with DC loaded by autologous or variant tumor antigen,and single culture CIK for autologous renal carcinoma cell,discuss the differences of immunocells for RCC immunotherapy,and provide a new research method for the patients who can't have an operation or get autologous tumor antigen.Methods1.Renal cell carcinoma cell primary culture and tumor antigen preparationUtilize the culture methods of explant tissue culture adherence,mechanical dispersion,enzymes digestion to establish short-term renal cell line,compare these methods and optimize.With ultrasonic cell disruptor to obtain autologous renal tumor antigen and renal cell carcinoma cell line 769-P whole-cell antigen.2.DC and CIK culture in vitroGather peripheral blood of patients or volunteers,use Ficoll-Paque Plus density gradient centrifugation to get peripheral blood mononuclear cells(PBMC),then add cytokines to induce DC and CIK.By day 8,harvest DC and CIK to co-culture,there are 3 groups in our experiment,DC without tumor antigen group,DC with autologous tumor antigen group,DC with variant tumor antigen group.We detect CD83,CD80, CD86,HLA-DR of DC,CD3,CD4,CD8,CD56 of CIK by flow cytometry.3.DC and CIK culture specific kill effect detection With CCK-8 kit detect the kill activity of CIK,CIK-DC,CIK-DC-autologous tumor antigen,CIK-DC-variant tumor antigen to the autologous renal cell carcinoma primary culture cell and cell line 769-P.Results1.Renal cell carcinoma cell primary culture and tumor antigenpreparationCompare these three methods,we find that enzymes digestion is the best,all the erperiment take on the third generation of primary culture short-term renal cell line. 100mg renal tumor tissue could extract 30～50mg whole-cell tumor antigen,and 1～3×10~7 RCC cells could extract 60～80mg tumor antigen.2.DC and CIK culture in vitroWith adding GM-CSF,by day 7,We could obtain the number of DC of 5～10%PBMC.After adding tumor cell lysate and cytokine,the mature markers of DC increase,such as CD83,CD80,CD86,HLA-DR.The markers of CIK that co-cultured with DC are higher than that single cultured CIK,such as CD3~+,CD3~+ CD4~+, CD3~+CD56~+,CD3~+CD8~+.3.DC and CIK culture specific kill effect detectionThe reaults show that the activity of CIK-DC-autologous tumor antigen group and CIK-DC-variant tumor antigen group are higher than that of other two groups,but there is no difference between them,CIK-DC group is the second,and the CIK group is the lowest.There is a positive correlation.The kill activity of patients' peripheral blood is lower than that of health volunteers',and there is significance.The activity of autologous peripheral blood to autologous tumor is equal to that of variant health volunteer,besides,there is significance between every two experimental groups (P<0.05).Conclusion1.DC induced by autologous or variant tumor antigen co-culture with CIK,can improve CIK in immunophenotype,generation and kill activity. 2.After induced by autologous or variant tumor antigen,DC has improvement in immunophenotype and generation.CIK co-culture with DC induced by tumor antigen has higher kill activity than that of other two groups.3.CIK co-cultured with DC has higher kill activity than that of single cultured CIK,this may provide an evidence for choosing clinical biotherapy.