Construction Of Tumor Vaccines By Gene Transfer Of T-bet Into Human Hepatocellular Carcinoma Cells And The Study Of Its Immunological Effect In Vitro

AIM:Th1 shift to Th2 plays an important role in tumor immunological tolerance. Raising the quantity of Thl cells will help to recover the ratio of Th1/Th2 of tumor patient and it is good to reinforce the cellular immune function. T-bet is the specific transcription factor of Thl ceils, it can regulate the differentiation of Thl cells. How T-bet gene can regulate the dominance differentiation of Thl cells and raise its efficiancy of antitumor immunity will be discussed by studying the antitumor effects of gene modified tumor vaccine. So high performance and more specific immune competent cells for active immunity of malignant tumor and new methods of prevention and treatment of tumor will be offered.METHODS:1. A pair of specific primers with restriction enzyme site of BglⅡand SalⅠwere designed and synthesized; according to gene sequence of human T-bet cDNA of Genbank. The total RNA was isolated from PBMC and the T-bet cDNA was amplified from the mRNA by RT-PCR, and then the PCR products of T-bet was inserted into pMD18-T vector and being sequenced. The pMD18-T-bet was double enzyme digested by BglⅡand SalⅠand the T-bet segment was extracted by agarose electrophoresis gel. Then the T-bet gene was inserted into pEGFP-C 1 to form the recombinant expression vector, pEGFP-C1-T-bet.The inserted segment was performed analysis and validation with double enzyme digestion and PCR.2. After 24h of transferring of pEGFP-C1-T-bet on human hepatacellular carcinoma HepG2 cell lines by lipofectamineTM 2000, the expression of GFP protein and T-bet was detected by fluorescence microscopy. The positive clone cells were screened out by G418. Then human hepatacellular carcinoma cell line HepG2-T-bet which stable express T-bet was established. The expression of mRNA and protein of T-bet in HepG2-T-bet was detected by RT-PCR and Western Blot, respectively. HepG2-T-bet cells were made into tumor vaccines TCV-T-bet by mitomycin C (MMC). The proliferation activity of HepG2-T-bet treated with MMC was examined by MTT and the expression of T-bet in TCV-T-bet was detected by RT-PCR6h, 24h and 48h before and after using MMC.3. Culture PBL of hepatocellular carcinoma patient in vitro, detect the levels of IFN-γ and IL-4 in supernatant by ELASA after stimulated by tumor vaccines and detect the phenotype of PBL by FACS. The PBLs were tested for cytotoxic activity against HepG2 cells by MTT after stimulated by tumor vaccines in vitro.RESULTS:1. Gel electrophoresis result of product of RT-PCR showed that specific proliferative band with relative molecular weight of 1608 bp appeared in the prospective place. The coding sequence of T-bet was confirmed by sequencing and that is identical with the sequence of human T-bet mRNA in Genebank. The inserted fragment was confirmed by double enzyme digestion and PCR analysis.2. Green fluorescence protein was expressed in human hepatocellular carcinoma HepG2 cells that transferred with pEGFP-C1-T-bet. After using G418 (640mg/L) for 14 days, the positive clone cells, HepG2-T-bet, which express T-bet stably were screened out. A 202bp fragment of RT-PCRproduct was detected by using the T-bet specific primers. The protein of T-bet, about 53kd, was detected by Western Blot. There was no proliferation activity of HepG2-T-bet after handling with MMC by MTT and the stable expression of T-bet in TCV-T-bet was detected by RT-PCR 6h, 24h and 48h before and after using MMC.3. (1) The levels of IFN-γin the lymphocyte and TCV-T-bet group after incubation for 24h,48h and 72h are significant higher than those in normal control group and lymphocyte and TCV-HepG2 group (p＜0.01) while the levels of IL-4 have no significant difference with other two groups (p＞0.05). (2) The levels of Th1 cytokine IFN-γin the lymphocyte and TCV-T-bet group are significant higher than those in normal control group ariel lymphocyte and TCV-HepG2 group (p＜0.05) while the levels of Th2 cytokine IL-4 are significant lower than those in other two groups (p＜0.05). (3) The lymphocytes'cytotoxic activity against HepG2 cells is (30.7±0.3)%; the TCV-HepG2 group is (14.1±017)%; while the nomal control is (7.4±0.5) %. Analysis of variance table shows that there is a significant diffenrence between the three groups (p＜0.05).CONCLUSION:The recombinant eukaryotic expression vector pEGFP-C1-T-bet was successfully constructed following successful amplification of complete T-bet gene. Human hepatacellular carcinoma cell line HepG2-T-bet which stable express T-bet was established. Human hepatacellular carcinoma vaccine TCV-T-bet was successfully constructed and the vaccine is effective in inducing antitumor immune response in vitro. We hope it could be a basis of tumor immunity and cancer gene therapy.