The Anti-tumor Effects Induced By Dendritic Cells Transfected With Total RNA From Murine Gastric Carcinoma Cells With 4-1BBL Gene

Objective: Gastric cancer is one of the most common malignant tumors in China. In recent years,the incidence is rising,the disease tends to younger,comprehensive treatment of advanced gastric cancer is poor. The one cause of difficult to cure Malignant tumors relates to tumor immune escape. Anti-tumor immunity is T-cell-mediated cytoimmunity.The activation of T cell needs double signals stimulus.The second signal is provided by costimulatory molecules on antigen presenting cell and corresponding ligand on T cell. The absence of the second dignal has been shown to lead to T cell anergy. A lot of studies show that tumor cell can escape body immune by absence of costimulatory.Dendritic cells is the most powerful antigen presenting cells,it can induces and maintains the initial immune response in vivo and vitro. DCs are uniquely potent in their ability to capture,process and present antigens to T cells. Now many investigators concentrate upon DC vaccine loaded whole tumor imformation to prevent immune evasion caused by antigenic wariation.4-1BB/4-1BBL is the other couple important costimulatory signal besides CD28/B7 in immune response and tumor immunity.4-1BBL belongs to ultra- family of tumor necrosis factor (TNF) and activates resting T cells separately or in coordination with CD28. 4-1BBL exists on the cell membrane superficial, and it expresseson activated T cell and B cell, monocyt, and dendric cell,neuro blastoma and some tumor cell. It plays an important role that maintains existen ce and memory response of T lymphocytes.4-1BB/4-1BBL may provide a new biological therapy of cancer.This study was that the co-stimulatory molecule 4-1BBL gene was transfected into gastric cancer cells,then the total RNA of gastric cancer cells transfected DC,production of tumor vaccines,to observe the anti-tumor effect in vitro.As for clinical immunotherapy of gastric cancer to lay the theoretical foundation for further.Methods:1 Insert the liposome-mediated pMKITneo/4-1BBL gene into the gastric cancer cells of the 615 mice,the positive cloning then turn into MFC/4-1BBL cell through G418 select.2 Assay the existence of 4-1BBL gene mRNA in the transfectional cell with reverse transcription-PCR(RT-PCR).3 Seperate the dendritic cells from 615 mice's myeloid tissue,then culture in vitro to increase the quantity and promote maturation.4 Wrap up the total RNA of mfc/4-1bbl cell with liposome and transfectouse dendritic cells to make the DC/4-1BBL/RNA vaccine.5 Splenic lymphocytes of the same race were harvested from mice and co-cultured with DCs and MFC/4-1BBL/DCs of inactivation.The proliferation rate of T cells was calculated by MTT.DCs ,MFC/4-1BBL/DC cells and T cel- ls were mixed and cultured.The kill rate of tumor cells was calculated by MTT. Cytokines were released in supernatants were measured for bioactive IL-12 and IFN-γby ELISA kits.All statistical analyses were carried out with SPSS 13.0 and P<0.05 were considered significant.Results:1 The expression of 4-1BBL mRNA by RT-PCR and cell shape observation of cell selecting after 4-1BBL gene transfered into MFC cellsWe got positive colone called MFC/4-1BBL after 4-1BBL gene transfered into 615 mouse cells MFC by the method of liposomes and selected by G418.Total RNA was extracted from MFC/4-1BBL and MFC cells.The cDNA was syntheted by reverse transcription.616bp specific strap was gotten by PCR amplification with mouse 4-1BBL specific primers in MFC/4-1BBL cells which indicated 4-1BBL mRNA was expressed stronger in this cell line.2 To observe cells under inverted microscope Cells number increased and the cell clusters were generated in third day of culture. DCs were suspension-growth and generated lots of cell processes and spiculated in fifth day of culture. Transferred DCs after 12 hours recovered virgin appearance.3 Mixed lymphocyte reacionT cells cycling were more efficiently activated by MFC/4-1BBL/DC(0.093±0.012,0.187±0.013,0.187±0.013)than DC(0.060±0.014,0.148±0.017,0.195±0.0 15)at the indicated stimulation to responding ratio (1:20,1:10,1:5).4 Cytotoxicity assaysTumor cells were more efficiently killed by T cells of MFC/4-1BBL/DC gro up(0.533±0.092,0.321±0.018,0.218±0.025) than DC group(0.342±0.023,0.267±0.0243,0.147±0.031)at the indicated effector:target(1:20,1:10,1:5).5 Cytokines5.1 The level of cell factor IL-12secreteELISA analysis showed that IL-12 secrete from high to low:MFC/4-1BBL/ DC(20.240±2.494pg/ml),DC(17.088±3.933pg/ml),immature DC(10.288±2.39 0pg/ml).5.2 The level of cell factor IFN-γsecreteELISA analysis showed that IFN-γsecrete from high to low: MFC/4-1BBL/ DC(9.451±2.925 pg/ml), DC(5.979±1.639pg/ml)Conclusion:1 The 4-1BBL gene is transfered into MFC cells by liposomes and this gene expresses stably. The DC vaccine has the stable activity after transferred by the total RNA of 4-1BBL/MFC.2 T cell proliferation are more efficiently activated by MFC/4-1BBL/DCs than DCs.IL-12 of transfected DCs are higher than other DCs in supernatants. The vaccine shows that the proliferation of T cell and body's immunity can be enhanced.3 Tumor killing ability to activate specific T-cell of MFC/4-1BBL/DC group are higher than untransfected DCs group and IFN-γin supernatants cultured with T cells are higher than untransfected DCs in supernatants.The vaccine shows that MFC/4-1BBL/DC vaccine can promote the anti-tumor activity.