| PURPOSE:Tlymphocyte immune responses play an important role in controlling tumor growth. Before T Lymphocyte play antitumor effects, i. e. secretion of cytokines or direct killing, they must first undergo activation. Optimal and efficient antigen presentation to lymphocytes is conducted through professional antigen-presenting cells (APC). Dendritic cells (DC) is the most powerful APC, which can markedly increase the antigen-presentation capacity by maximizing the pepitide-MHC complexes on the cell surface and upregulating the co-stimulatory ligands B7-1 and B7-2, adhesion moleculees such as IL-12 that promote full activation of lymphocytes. Full activation of antigen-specific T cells requires two signals-one signal coming via the TCR and the other signal throughengagment of co-stimulatary molecules. T cells receiving one signal via their TCR are turned off by MHC(major histocompatibility complex), via T cell CD28 binding to B7 on the DC induce Tlymphokine and T cell proliferatiion. DCs play a pivotal role in the development of antitumor immunity.T lymphocytes immune response induced by direct antigen-pulsed DCs can not maintain longer for peptides-MHC department or MHC molecules degeneration. The method of DCs loading with tumor antigen gene is another potential approach to enhancing and maintaining immune response. Carcinoembryonic antigen (CEA) is amuch studied tumor associated antigen. It is highly expressed by 90 %gastrointestinal,50% breast and 70%lung adenocaicinomas. CEA gene was transferred into human DCs, and specific anti-cancer effecs induced by the vaccine was observed. This test is part of my tutor's.Hang you-tian has observed the induction of crcinembryonic antigen (CEA)-specific cytotoxic T-lymphocyte responses in vitro when he transfected DCs with pcDNA3-CEA, and has observed the immunity effects of the DCs(pCEA) inoculateing against to CT 26 (hCEA+) loaded in BALB/C mice.After vaccination with the CEA gene -modified DC, the survival time of the mice vaccinated with CT26+(CEA+) ws prolonged more potently than that of the mice vaccinatd with other DCs.OBJECTIVESFirstly To prepare the vaccine of human DCs and tumor cells transfectde with pcDNA3-CEA, and to observe the induction of carcinoembryonic antigen (CEA)-specific cytotoxic T-lymphocyte responses in vitro. Secondly , to observd the immunity effects of the DCs(pCEA)inoculating against to tumors(hCEA+) . METHODS1 In intro preparation human Mode and tumor cellThe peripheral blood mononclear cells (PBMC) were isolated from human fresh peripheral blood. PBMC were cultured in complete medium and supplemented with 1000U/ml rhGM-CSF and 500U/ml rhIL-4, MoDC were collected on day 9.Tumor cells were cultued with 10 % complete medium.2.The plasimd encoding human carcinoembryonic antigen was transfected with lipofectine into human DCs and human tumor cells such as MGC803, LS174T and Eel09. The culture supernatants were collected for assaying carcinoembryonic antigen by.3. Amplification of humancarcinoembryonic antigen gene by RT-PCR.Total RNA were isolated from DCsand tumor cellsand were reverse transcriptd into cDNA by AMV reverse transcriptase. The C-sequence of collagen encoding CEA were amplifed by PCR,4.Cytotoxicity(CTL) assy in vitro :on days autologous lymphocytes were used as effector cells, and cocultured with MGC803, LS174T and Ecl09 cells: target ratio of 10:1, 20:1, 40:1, then DCs and DCs(pCEA) were adds as stimulators :effector tatio of 1:10, There kind of cells were cocultured in 96 flat-bottom microphates for 72h, then cytotoxicity of lymphocytes was analyzed by MTT assay.RESULTS1. The plasmid with human CEA DNA was introduced into human DCS by lipofectine We successfully detected CEA mRNA expression in DCs(pcDNA3-CEA+) with RT-PCR. CEA expression level in the DC (pcDNA3-CEA+) culture supernatant is higher than in the DCs culture supernatant.2. The plasmid with human CEA DNA was introduced into MGC803, LS174T, EC 109 by lipofectine We successfully detected CEA mRNA expression in MGC803,LS174...
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