Dendritic Cells Transfected With Tumor RNA For The Induction Of Antitumor CTL Against Protate Cancer

Objective:To investigate the effect of dendritic cells (DCs) transfected with tumor total RNA,and to investigate whether bone marrow-derived dendritic cells transfected with tumor RNA induce specific CTL against prostate cancer ex vivo.Methods:1.prostate cancer cell culture.RM-1 cells was cultured and and collected in exponential phase of growth to prepare for next experiment.2. Culture and identification of mouse bone marrow derived DCs.Mononuclear cells isolated from mouse bone marrow were induced into dendritic cells by being cultured in GM-CSF and IL-4, and then examined from aspects of morphology, phenotype and function of stimulate T lymphocytes.3. DC transfected with tumor RNA and induce specific CTL.Total RNA was isolated from mouse prostate cancer cell carcinoma cell RM-1 cells by Trizol.After culture for 6 day, DCs were pulsed with total RNA from RM-1 cell by liposome transfection. The expression of cell surface molecules were detected using flow cytometry before and after tranfection. Specific cytotoxic T lymphocyte (CTLs) induced by DCs transfected with tumor RNA was assessed with LDH.Results:Modality of mononuclear cells diversified after being cultured in GM-CSF and IL-4 for 3 days, and growthing clustered-liked. When culturing for 5 days, typical morphology with dendritic processes can be observed. A large number of morphologically typical dendritic cells were observed after the culturing for 8 days, under scan electrom microscope, DCs displayed typical morphology with dendritic processes and high expressed specific marker of bone marrow derived DC-CD11c and costimulatory molecules CD40,CD80,CD86,and MHC-II, mature BMDC could stimulate syngenic and allogenic mixed lymphocyte proliferation.. Compared with non-transfected DCs, costimulatory molecules CD40,CD80,CD86,and MHC-Ⅱhigher expressed on DCs transfected with tumor total RNA.CTLs induced by DCs tranfected with RM-1 cell total RNA could specificitily kill RM-1 cells,while no significant effect against control cell-mouse liver carcinomar cell Hepa-1 was seen. IFN-γproduction of CTL induced by RNA-DC was increased significantly than imDC and untransfected DC. RNA-DC transfectd by induced higher CTL cytotoxicity against RM-1 cell than imDC and untransfected DC. (p<0.01) Conclusion:A large number of dendritic cells can be generated by culturing the mononuclear cells derived from mouse bone marrow in vitro, high purity imDC can be acquired when culturing for 6 days, which possess the potential ability to induce specific CTL after pulsing with tumor antigen.RNA transfection has no effect on surface antigen's expression of DC.DC transfected with mouse prostate cancer cell total RNA could stimulate exprssion of costimulatory molecules,induce specific CTL against prostate cancer cell and thus will lay a foundation for future research in producing the anti-tumor vaccine.