| Colorectal cancer is one of the commonest tumors in our country.There is a increase trend in disease rate. Yet in spite of extensive efforts over the last 50 years ,only modest improvements in overall survival of patients with this disease have been accomplished through refinements of surgery,chemotherapy and radiotherapy. Some 50% of affected patients die in recurrence or metastases.In order to improve survival and heighten the patients's own immune response to cancer,the development of cancer -specific vaccines become a key approach to immunotherapy.The dendritic cell(DC) is now recognized to be the most effective antigen-presenting cells .Tumor-induced dyfunction of DC is a mechanism of escape immunosurveillance.Thus, the study on immune therapy is focused on enhancing functional activity of DC and breaking tumor escape mechanisms. Tumor lysate-pulsed DC associated with systemic administration of interleukin 2 was used in our experiment to therapy mice colon tumor model in order to investigate its anti-tumor mechanisms and provide preclinical rationale experimental theory. Materials:l.FemaleBALB/C(H-2d)mice,6-8-wk-old,18-22g. 2.C26 cell line is a murine colon adenocarcinoma cell line derived from BALB/C(H-2d) mice;B16 cell line is a murine melanoma cell line derived from C57BL/6(H-2b) mice. Methods:1. Preparation and identification of DC:Bone marrow cell suspensions were prepared with aseptic technique,and erythrocytes were removed.The murine bone marrow-derived precursors were induced in vitro culture with both granulocyte-macrophage colony-stimulatin factor(GM-CSF) and interleukin-4(IL-4).On day 7,DC were harvested and examined by hematoxylin and eosin stain technology and flow cytometer.2. Preparation of C26 tumor lysate-pulsed DC :DC were incubated with freeze-thawed repeatedly C26 tumor lysates for 16 hours.3.T cell proliferative assay(allogenic mixed lymphocyte reactions)in vitro:Bearing-mice splenic T cell suspensions were prepared with aseptic technique. The responder T cells were cocultured with either C26 tumor lysate-pulsed DC in combination with 500u/mlrhIL-2,unpulsed DC aloneJDC pulsed with C26 tumor lysate were determined at a stiniulator-to-responder ratio of 1:100,1:330,1:1000 by 3H-TdR incorporation assays .Then Cpms were obtained.4. Cytotoxicity assay in vitro:Tumor lysate-pulsed DC induced splenic cytotoxic T lymphocyte activity. The percent specific lysis of C26 and B16 was calculated at tfieeffector-to-target ratios 50: 1, 25: 1, 12.5: 1 with MTT method.5. Tumor model and in vivo experiments:In vivo,BALB/C mice were inoculated subcutaneously in the rightjflank with lx!05CT26.Then, tumor-bearing mice were randomly divided into four groups and contained 7 tumor-bearing mice for each group .Animals subsequenting bearing 1 day were treated with four-weekinjections s.c of 1xl06 C26 tumor lysate-pulsed DC in the right inguen.IL-2 was given s.c.daily at 10,000U at peritumoral site for 5 consecutive days after each immunization. Mice of control group received both no treatment, C26 tumor lysate-pulsed DC alone,or IL-2 alone.The tumor growth were monitored at 2-day interval since day 11 and the survival time of mice of each group was recorded . Results: 1 .Identification of DC:The cells showed branching-like and pseudopod-like under inverted light microscope.The bias nucleus of DC were basophilic after HE staining. These DC belonged to myeloid lineage of DC(CDllc+)and high express MHCU(Ia)and co-stimulatory molecules(CD40,CD80,CD86)by FACS. 2.Proliferative assay:T cells with C26 tumor lysate-pulsed DC resulted in a significant proliferative response compared with tumor lysate-unpulsed DC and high 3H-TdR incorporation with elevating responder-to-stimulator ratio.IL-2 promoted T cells growth.There were significant difference between IL-2 group and IL-2 unenrolled group by LSD test(P<0.05). 3.Cytotoxicity assay in vitro:The percent specific lysis of C26 tumor cells was68.84%,58.36...
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