Induction Of Antitumor Immunity In Vitro Using Human Dendritic Cells Pulsed With MAGE-3 MRNA As A Tumor Viccine

Malignant tumor is a very common disease which threatened people's health seriously. Immunotherapy has been the major therapeutic strategy in cancer treatment. The key of cancer immunotherapy is to enhence the immunogenicity of tumor in order to induce specific antitumor immune response, as the strategy of tumor vaccine. MAGE is a malignant melonoma gene family, its gene members are expressed in multiple malignant tumors but not in normal tissues except testis and placent. Combining with HLA molecules, peptides encoded by MAGE genes become peptide-HLA compounds and identified by TCR of CTLs, then induce specific cytotoxicity. Therefore MAGE antigens are considered as suitable immunogens for cancer immunotherapy and for tumor vaccines. Nucleic acid vaccines, comparing with the previous vaccines, are more safe and more efficient especially for RNA vaccines. Dendritic cells (DC), as natural adjuvants, are the most potent professional antigen processing cells (APC) in human. Many researches proved that tumor vaccinations based on DCs can induce more potent antitumor specific cytotoxicity. In our study, we constructed a tumor nucleic acid vaccine using DC loaded by MAGE-3 mRNA, and evaluated its antitumor efficacy in vitro. To our knowledge, this is the first study that using MAGEmRNA pulsed DCs as tumor vaccine.Parti Expresion of multiple MAGE genes in gastric cancers and itscorrelations with clinical characteristics1. Detection of MAGE-1, -2, -3 genes in gastric cancers and normaltissuesWe detected the expression of MAGE-1, -2,-3 genes in 62 gastric cancer tissues and corresponding normal tissues by RT-PCR method. The expression rate of MAGE-l, -2, -3 genes in gastric cancer tissues were 11.29% (7/62) ,24.19% (15/62) and 38.71% (24/62) respectively, while none of MAGE genes was detected in corresponding normal tissues. 50.0% gastric cancer tissues expressed at least of those 3 MAGE genes. According to the statistical analysis, the expression rate of MAGE-3 gene was significant higher than that of MAGE-1 and MAGE-2 gene in gastric cancers (p<0.05).2. Analysis of the correlation between MAGE-1, -2, -3 gene expressions and clinical characteristics of gastric cancersBy statistical analysis, we found that there were no correlations between the expressions of MAGE-1, -2, -3 gene in cancer tissues and age, gender, depth of tumor invasiveness, lymph nodes involvement and distal metastasis of gastric cancer patients (p>0.05).We concluded that MAGE-3 gene was relatively high expressed in gastric cancers, its expression had no correlation with the clinical characteristics of gastric cancers. As a tumor specific antigen, MAGE-3 is a ideal immunogen for antitumor specific immunotherapy, and can be treated as a target antigen of tumor vaccines.Part IIConstruction of tumor viccine using dendritic cells pulsedwith MAGE-3 mRNApMAGE-3 ORF/T as a carrier and pCI as a standard plasmid were cut by restriction endonuclease Mlu I and Sal I respectively, MAGE-3 ORF and linear plasmid pCI were obtained with sticky ends either, then they were linked by T4 DNA ligase. After screening and sequencing the recons, we got the stable expressing vector of pCI-MAGE-3. The recombinant plasmids were transferred to E.coli and amplified. By plasmids extration, endonuclease digestion and purification, MAGE-3 cDNA templates were obtained. After in vitro transcription with T7 RNA polymerase and m7G Cap analog, finally we got the capped MAGE-3 mRNA with poly(A) tails. Using same method we constructed recombinant plasmids pCI-EGFP and acquired EGFP mRNA meanwhile, as the control group and irrespective antigen.Peripheral blood mononuclear cells (PBMC) obtained from venous blood of cancer patients were isolated by Ficoll density centrifugation. PBMC were cultured in RPMI-1640 medium supplement with 10% heat-inactivated human serum for 2 hours. The adherent cells were then cultured in the same medium as last step in the presence of rhGM-CSF and rhIL-4 for 5 days as immature DCs for transfection. The non-adh...