| Background and objectiveThe immunosurveillance effect can help the body to recognize specific tumor cells and eliminate them effectively, which has obvious curative effect especially on early spreading tumor foci. However, with in vivo progression and development of tumors the immunosurveillance effect can mostly be induced into immune tolerance when the immune system can not recognize and kill tumors cells so that the tumors cells could continuously proliferate and metastasize.Th1/Th2 differentiation involved in the processing. Therefore it can help to deeply understand the mechanisms of tumor development and progression and offer us more scientific and healthy treatment measures to further find out and use immunotherapy. CCL2, also named after macrophage-chemoattractant protein-1(MCP-1) , is a member of C-C family chemokine and it is a powerful chemoattactant for leukocyte with its specific receptor such as monocyte/macrophage, nature killer(NK) cells and eosinophils when pathogen invades our body or neoplastic cells develops in some tissue. There is ample evidence of chemokines playing a role in decreasing tumor growth and removing metastasis of neoplastic cells.. Most previous reports mentioned that CCL2 participates in anti-tumor activities with macrophage and non-specific celluar immunology and the flux of macrophages attempted to destroy the tumor tissue as well to repair the damages with secreting some growth factor,e.g. VEGF, TGF-β and angiogenesis. CCL2 is a chemoattractant for a wide variety of leucocytes except for monocyte/macrophage, including T lymphocytes. More recent studies have begun to elucidate the role of endogenous CCL2 in the development of the Th2-type and Th1-type immunity in some inflammatory reactions. Joerg et.al. have demonstratedthat Th2 cells may rapidly eliminate lung metastasis and act independently of cellular immunity. It was also suggested that development of Th2 immunity could promote the sensitivity of tumor-specific antigens and result in eosinophil-induced tumor regression, which might provide a reliable approach of tumor immunotherapy. Based on previous studies, our experiment was to constitute recombinant mouse melanoma B-16 cells expressing CCL2 and to restrain the metastasis and growth of tumors by highly expressed CCL2. Furthermore, we aimed to validate the differentiation tendency of Th cells in the microenvironment with highly-expressed CCL2 and analyze the control function of Th2 cells differentiation on tumor growth and metatstasis. At last, we can analyze the important role of chemokine CCL2 played on tumor immune response, and provide theoretical foundation for treating tumors by CCL2, and find a new way to modulate the balance between tumor immunosurveillance and immune evasion.Part I Recombination of murine CCL2 in melanoma B-16 cells and identification of its biological functionMethods: (1) Recombination of murine CCL2 in melanoma B-16 cells Total RNA was abstracted from murine actively macrophage in abdomen. The cDNA coding for the full-length CCL2 was amplificated from mRNA of activated celiac cells by Reserved Transcription Polymerase chain reaction, then it was subcloned into the eukaryotic expression vector pcDNA3.0. Transfection with pcDNA3-CCL2 and a control vector pcDNA3.0 was performed with Fungene-6. The transfected cells were survival and expanding in presence of Genetacin. The clones(A4, A5, A6, Bl) transfected with CCL2(CCL2-B-16) and 2 ones (H6,I5)with control vector(mock B-16) were selected for further study because of their vigorous proliferative capacity. (2) Detection of expression of CCL2. Total RNA were abstracted from cells by guanidinium thiocyanate methods and Reserved Transcription PCR was performed. Gray scale ratio of CCL2 to G3PDH DNA strap was mean of 5 above-mentioned reactions. To measure the production accurately the amounts of CCL2 were detected by ELISA. (3) Detection of Bioactivity of CCL2 Bioactivity of CCL2 expressed bytransfected cells was evaluated by measuring the chemotaxis of active marcophage of mice. The distance between mid holes and above or low one was measured using ocular micrometer and the chemotaxis index was quotient of distance in CCL2 holes to the other one. Results: (1) Level of CCL2 was measured in CCL2-B16 cells, Mock -B16 cells and WT-B16 cells before clonal selection. By the method of Reserved Transcription PCR and ELISA it could be infer that the production in A5 clonal was a stable and high one. (2) Means of migration distance by CCL2-B16 was 1.4 mm and the means by WT-B16 cells was 0.27, so the chemotaxis index was gained as 5.2. It was demonstrated that CCL2-B16 melanoma were harvest with high production and chemotaxis function and A5 clones would be used afterward. Conclusion: The strains for high level of CCL2 in melanoma B16 cells have developed and could secret chemokine with chemotaxis function.Part II The differention of thymocytes by high-level combination CCL2 B-16 melanoma in vitroMethods: Primary thymocyte were gathered from thymus gland by the methods of digestion with 0.25% trypsin-EDTA. The 2X 105 B-16 melanoma(each well in each types) were seeded at 2X107 per well onto the inner surface then thymocytes were added into the plates with melanoma B16 cells con-culture.6h later, thymocyte were centrifiigated from harvested cultural suspension. Then total RNA were abstracted from the cells.To measure the cytokines in transcription related to Thl or Th2 cells, RT-PCR reactions were proceeding just as.IL-2, IFN- Y (Thl dominant),.IL-4, IL-5, IL-10(Th2 dominant) and inner stander G3PDH just as above. The same 5 trails were replicated and the result were analysis as one described above. The level of STAT-6 from 4d concultured thymocytes were examined by methods of western-blotting Results: After 6h con-culture thymocytes were gathered and by method of Reserved Transcription PCR IL-4, IL-5 and IL-10 could show extent of Th2 cells polarization and IL-2 and IFN-y could represent development of Thl immunity. Ratio of cytokines mentioned above to inner reference G3PDH were obtained and mRNA reflecting Th2 cells was far more than one for Thl cells. Also the level of STAT-6 in CCL2 groupwas far more than others. This provides a direct proof to explain that CCL2 could induce Th2 polarization in microenviroment. Conclusion; The expression of CCL2 can induce thymus cells to differentiate towards Th2 cells, while the differentiation degree of Thl cells among the groups make a few changes. It shows that CCL2 in tumor microenvironment can induce primary T cells to differentiate.Part DI Transfected CCL2 Releasing Tumor Progression with T helper 2 lymphocyte PolarizationMethods: (1) Animal models: WT-B16(Wild Type), CCL2-B16 and mock-B16 melanoma cell suspending were injected intravenously into recipient mice(5 in each group) to establish lung metastasis animal models. After 15d the mice were killed and lungs were harvest and were in the processing of H.E. staining. To observe the function of CCL2 in long animal models with local cancer growth 5><106 each type neoplastic cells were innoculated subcutaneously into the left hind leg and lOd afterwards the different cells(equally amount) were injected into the same mice at the right leg in the same way. Statistical analysis of survival curves was drawn using the Log-Rank test. (2) Immunohistochemistry: With immunocytochemistry methods, the expressions of CCL2 and IL-4, IFN-y could indicate the status of CD4+ T cells differentiation in CCL2 expression. (3) Flow cytometric analysis: The 3 mice injected intravenously were ready for following experiment 7d afterwards. The mononuclear leucocytes were harvested from albuguina layer(under plasma). Spleen was removed and incubated at 37*C. Then cell suspension immunobelled with designated mAbs then were in processing of Flow cytometric analysis. Results: (1) Eradicating the metastasis for being transfected CCL2: It was observed that lungs of wild type were coved with black tumor metastasis and transfection of B-16 melanoma with CCL2 could lead to a substantial reduction of metastasis.The survival curves indicate the beginning inoculation of CCL2 cells could promot the longevity no matter reinnoculation of WT cells. (2) Relationship of CCL2 and Th2 polarization in tumor microenviroment: hi tumor foci CCL2 had a high production in deeply brown where expression of IL-4 was conspicuous and expression of IFN-ywas insignificant. B-16 melanoma transfected with CCL2 could be required for Th2 polarization. Several typical section were chosen for detection CCL2 and IL-4 with two colors to find CD4+ Th cells status for tumor decreasing by CCL2. (3)Changes of T cells in blood by high level of CCL2: It seemed that CCL2 expression stimulate IL-4 production in mice injected tumor cells. FACS analysis interestingly told CD4+ positive cell number in CCL2-B16 group was more than one in WT-B16 group and CD8+ positive cell number differ tinily. Positive rate of CD45RO, expressed on memory T cells and/or activated T cells which had been stimulated by antigen, had been increased by transfection of CCL2-B16 melanoma. Conclusion: (1) Cytokines represented Th2 polarization had been increased in animal models inoculated with melanoma transfected with CCL2, just as well the quantity of CD4+ T cells in blood raised followed injection intravenously. However, there was no obvious difference of Thl related index( e.g. cytokines and CD8+ cells). It was demonstrated that CCL2 expression in melanoma could induce Th2 polarization in mice. (2) First injection of CCL2-B16 could inhibit proliferation of tumor cells, moreover, even if secondly injection WT-B16 on the animal, its life would not be shorten. It was suggested that CCL2 might have the ability of activating the T cells and make memory T cells more power accompany. (3) From our research it could be inferred that CCL2 might regulate the difference of specific T lymphocyte with inhibiting tumor growth anf metastasis.Conclusions1) We have shown that highly expressed CCL2 can induce Th cells to differentiate into Th2 cells in the tumor microenvironment by high expression of CCL2.2) It has been verified that the ability of tumor cells to express CCL2 determines the amount of memory T cells in vivo and dominates metastasis of tumors effectively.3) Three animal models was established to study the correlation between chemokine CCL2 and T cell differentiation, the metastasis ability of carcinoma. It was suggested that CCL2 could modulate the differentiation of T cells, resulting in inhibition of the progression and metastasis of tumors. However, CCL2 played its major role in... |
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