The Expression Of IGF1Rα In Mammalian Cells And It's Priming Specific CTL Against Hepatocellular Carcinoma Cell Line HepG2

Background and objective:Hepatocellular carcinoma(HCC) is a common malignancy with a very poor prognosis.Only a minority of patients are eligible for surgical therapies.Therefore,novel strategies to prevent proliferation of malignant cells are urgently needed.A promising approach may be the prophylactic vaccination directed against a tumor-associated antigen(TAA)or tumor-special antigen(TSA).IGF1Rαis usually expressed in high concentrations in the liver cancer,however, low expression in the normal liver cells.IGF1Rαmay be a target for special tumor vaccine against IGF1R in the special immunotherapy and gene therapy.Dendritic cells(DC) are considered the most potent antigen-presenting cells(APC) because they may incept and manage the antigen,and stimulate the primary activation of T cells causing to special immune reaction against the antigen.So DCs are promising new tools for the immunotherapy of cancer.Many research approved the DCs of the cancer patients get worse,and MHC,CD80,CD86 of DC expression decline.It may be a development aspect that TAA or TSA stimulating normal DC cells immune cancer patient as vaccine.The immunogen can be TAA,TSA,tumor lysates, DNA,mRNA,etc.The coculture of DC and TSA antigen is common method.Methods:1.distill RNA of HepG2,enlarge IGF1Rαantigen using RT-PCR, connect aim gene and pGEM-Teasy carrier,construct PcDNA3/HA and IGF1Rαrecombining carrier,instantaneous expression in the HER293 cells, inspect using Western Blot in the 72 hours.2.we induced peripheral blood mononuclear cell into DC cells with rhGM-CSF(1000 u/ml).rhIL-4(100 ng/ml). After 4 days of culture,DCs were harvested pulsing of autologous DC with IGF1Rα.Modality of DCs were observed bythe microscope.FACS analyses the phenotype of DC.After coculture of DC and T cells in 7days by 1:5, to measure cytotoxicity of CTL in MTT assays.Results:1.we obtain IGF1Rα-pGEMT carrier,and IGF1Rα-pcDNA3/HA expression carrier.We find the protein which molecular weight is 34KD after transfecting HEK293 cells.2.The matured DCs sticked out many pseudopods.By FCM analysis,the matured DCs' surface marker were upregulated compared with immature DCs,including CD86 87.9%,CD83 69.7%,CD80 88.69%,CD40 90.1%,but CD14 were downregulated which numerical value is only 11.8%.MTT assay revealed that the cytokine-driven DCs with antigen induced a strong specific CTL cytotoxicity to HepG2 campare with comparison groups(P＜0.05),The cytotoxicity rate 30.1%when the ratio of the effect cell to target cell was 20:1.The comparison groups has no distinct difference.The CTL cytotoxicity with IGF1Rαprotein loading DCs to HepG2 was distinct difference compare with to NIH3T3.Conclusion:The recombined human IGF1Rαprotein was expressed successfully in mammalian cells producing mature DC cells with its stimulation can induce specific HepG2 cells CTL.The results revealed that IGF1Rαis important for immunotherapy of HCC of human.