| Colon cancer is one of the commonest tumors in our country and there is an increase trend in the disease rate. We have made tremendous advances in the diagnosis and treatment of colon cancer, but it remains a high mortality and morbidity in recent years in china and it has endangered health and life of people greatly. Multidisciplinary treatment was applied to the patients with colon cancer while other treatment strategies for colon cancer should be searched after. Immunotherapy is deemed to be the fourth approach which had curative effect to colon cancer following surgical treatment, chemotherapy and radiotherapy at the present time and DC vaccine plays an important role in anti-tumor immunotherapy. With high security and accurate anti-tumor effect, DC vaccines may be an effective way for the treatment of tumor recurrence and metastasis.Tumor can express self antigen like normal cells, and can express aberrant or unique antigens which can be recognized by immune system which is the foundation of tumor immunotherapy. Dendritic cells, acting as professional and the most powerful antigen presenting cells, can effectively intake, process and present antigens, activate initial T cells, and thereby work as the center of immune responses. Much is known of their important role in anti-tumor, resisting injection, transplant repulsion and anti-self immune disease. So DC based cancer immunotherapy is one of important direction in tumor biologic therapy in the anti-tumor responses. In recent years, growing concern has been concentrated in DC-based tumor immunotherapy. Loaded with tumor antigens, DC can function as vaccines. And for DC vaccines having high efficiency in presenting antigen, they are becoming a representative of a new generation of vaccines.Currently, a small amount of DC cancer vaccines have been used in clinical practice, but the result was not satisfactory mainly due to DC in vitro failed to reach the ideal state of maturation. So they can not stimulate the body to produce an effective specific anti-tumor response. Toll-like receptors (TLRs) have recently emerged as important pattern recognition receptors (PRRs) in the innate immune system that detect microbial infection and trigger antimicrobial host defense responses. And they play a crucial role in the host defense against invading microorganisms by recognizing pathogen-associated molecular patterns (PAMPs). Toll-like receptors exist as highly conserved pathogen sensors through-out the animal kingdom and they represent a key family of molecules bridging the ancient innate and adaptive immune systems.Dendritic cells themselves are highly specialized subsets of cells with the major roles of antigen presentation and stimulation of lymphocytes. The dendritic cell functions of inducing immunity are regulated by their own activation status, which is governed by pattern recognition receptors, including Toll-like receptors, expressed at the surface and within the cytoplasm and endosomal membranes of dendritic cells. They can recognize foreign antigens, in turn, can stimulate DC maturation and thus induce specific immune response.Objective:To compare the immune functions of the dendritic cells (DC) activated by combined Toll-like receptor (TLR) ligands and DC generated in conventional way, to compare the therapeutic effects on colon cancer by cytotoxic T lymphocyte(CTL) induced by different DC in vitro, to observe the anti-tumor effect of DC vaccine activated by combined Toll-like receptor ligations on murine colon cancer model, and try to establish the basis for exploring the new tumor vaccination for immunotherapy.Methods:1. Human CEA transgenic C57BL/6 mice at the age of 6 to 8 weeks were used as the animal model in the experiment. The DC were activated by combined Toll-like receptor (TLR) ligands and in conventional way. In the sixth day,analyze the secretion of IL-12p70 in culture supernatants in different groups.2. DC were pulsed with mouse MHC class I-restricted CEA epitope (CEA526-533)acting as CEA specific DC cancer vaccines, and then cultured with mouse spleen cells as effector cells for a week. At last, we conduct the cytotoxicity assay to investigate their therapeutic effects against CEA-expressing colon cancer(MC38-CEA) and non-CEA-expressing colon cancer MC38 in vitro.3. Establish a mouse model of colon cancer for experiment in vivo. The growth status of the subcutaneous tumors was observed two times per week and the survival days of mice was recorded. Finally the mean tumor volume in each group was analyzed for plotted curves and survival curves of mice in each group was depicted. Results:1. Combined TLR activated DC secretion of IL-12 is significantly higher than conventional generated DC(P<0.01).2. The effector cells cultured with combined TLR activated DC c show significant cytolytic activity against MC38-CEA cells at high E:T ratios (P<0.05), and there is no remarkable cytolytic activity observed in other groups.3. To MC38 cells, the effector cells cultured with both combined TLR activated and conventional generated DC have no cytolytic effect, and there are no differences between them (P>0.05).4. In the mouse tumor model with MC38-CEA colon cancer cells, mean tumor volume in the group of TLR activated DC pulsed with CEA peptide is significantly smaller than that of other groups (P<0.01),and with longer survival days (P<0.01).5. In the mouse tumor model with MC38 colon cancer cells, mean tumor volume in all groups has no significant difference (P> 0.05), and the survival time in each group has no significant difference (P> 0.05).Conclusions:1. Combined TLR activated DC cancer vaccine can induce some CEA specific cytolytic activity against MC38-CEA cells in vitro cytotoxicity assay, however there is no remarkable cytolytic activity observed in conventional generated DC cancer vaccine immunized group.2. Combined TLR activated DC vaccine could inhibit the growth of MC38-CEA colon cancer cells and prolong survival days. |
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