Experimental Study On Il-2 Gene Transfection Of Cik Cells Combined With Dendritic Cells Against Hepatocellular Carcinoma

Objective:To Clone human Interleukin-2(IL-2)gene from Jurkat tumor cells and construct a recombinant adenovirus vector pAdBM5-GFP-IL-2.Methods:Total mRNA was extracted from Jurkat tumor cells stimulated with PHA,and specific primer pairs was designed in order to clone human interleukin-2(IL-2)gene by RT-PCR method.IL-2 gene was directional constructed into adenovirus vector(pAdBM5-GFP)after digestion by restrictive endoenzyme Bglâ…¡,and Pmeâ… .The constructed recombinant adenovirus vector was named pAdBM5-GFP-IL-2.The lined recombinant adenorirus vector and viral skeleton were cotransfected into HEK293A cells by coprecipitate of calcium phosphate.Recombinant adenovirus was amplificated and purificated in HEK293A cells.Recombinant adenovirus titer was finially tested by TCID₅₀.Results.IL-2 gene was cloned correctly and recombinant adenovirus shuttle vector pAdBM5-GFP-IL-2 was constructed successfully and the titer of recombinant adenovirus is as high as 5.0×10⁷pfu/ml.Conclusions" The constructed recombinant adenovirus vector pAdBM5-GFP-IL-2 would lay down the basis for further amplifying the related recombinant adenovirus and developing gene therapy of primary liver cancer. Objective:To study the effect of the cytokine induced killer cells infected by IL-2 co-cultured with dendritic cells on cytotoxicity against hepatocellular carcinoma cells.Methods:Peripheral blood mononuclear cells(PBMC)were isolated from healthy adult donors by density gradient centrifugation.After 2 hours incubation of PMBC,CIK was generated from non-adherent cells by anti-CD3McAb 50ng/ml,IL-2 1000U/ml,IL-1 1000U/ml and IFN-γ1000U/ml,And DC was induced from adherent cells by rhIL-4 100ng/ml and rhGM-CSF 100ng/ml. Recombinant adenovirus vector pAdBM5-GFP-IL-2 infected CIK at 100 MOI generated CIKpIL-2,DC cells were pulsed with HepG2 lysate at a ration of 1:3 and generate DC-Ag.Mature DC was harvested after 9 days incubation.DC and DC-Ag co-cultured with CIK and CIKpIL-2 at a ration of 1:3.CIK cells,DC-CIK cells,DC-Ag-CIKcells,DC-Ag-CIKpIL-2 cells,DC-CIKpIL-2 cells as effective cells killed the HepG2 cells.Effectors:target cells(HepG2)ratio were 10:1,20:1,40:1,The cytotoxicity activity was investigated in MTT assays against HepG2 hepatocellular carcinoma cell line,K562 and H7404 cell lines were cultured as the cytotoxicity control.Results:On comparison with non-transfected CIK cells co-cultured with DC,transfected CIK cells co-cultured with DC had a significantly higer lytic activeity against HepG2 cells,a human Hepatocellular Carcinoma Cells.Lytic activity of the transfected CIK cells co-cultured with DCs pulsed with tumor lysate had not difference from the transfected CIK cells co-cultured with DCs without pulsing.Conclusions:Cytokine Induced Killer Cells infected by IL-2 had high lytic activity against HepG2 HCC cell line,which were enhanced by co-culture with DC,It plays an important role for the adopted immunotherapy of HCC.