| Objective: The interaction between tumor cells and tumor stroma has been reported to play an important role in tumor growth and intrusion. Interactions between the tumor cells and their microenvironments lead to extracellular acidosis in solid tumors. In the presence of tumor microenvironment, macrophages produce immunosuppressive cytokines, key tumor-promoting molecules, and some inflammatory cytokines. Mononuclear macrophages also have heterogeneity and play important roles in the biotherapy of tumors. Therefore, it is of significance to modulate the function of these cells in acid microenvironment in the biotherapy of tumors. In this study, the biological behavior of mononuclear macrophages and Tca-8113 cells were investigated by changing the p H value of cell culture fluid, and the effect of Triepoxide(TP) on the function of mononuclear macrophages in acid microenvironment was investigated in vitro experiments, with a view to providing more experimental data for developing anti-tumor drugs from Tripterygium wilfordii Hook. Methods: 1. Tca-8113 cells were cultured under acid microenvironment(p H 6.6, p H 6.8,p H 7.0)and normal microenvironment(p H 7.2) for 64 h. The contents of IL-8, MCP-1, and HIF-1 in each supernatant were measured by ELISA assay. The number of Tca-8113 cells was subsequently determined by celltiter 96® aqueous one solution cell proliferation assay(MTS). 2. Mononuclear cells separate from a healthy person’s peripheral blood were cultured under acid(p H 6.6, p H 6.8, p H 7.0) microenvironment and normal microenvironment(p H 7.2) with Tca-8113 cells or not for 64 h which was determined by preliminary experiments. The expressions of CD68, CDl63 and CD80 on the surface of mononuclear macrophages were detected by flow cytometry. The contents of IL-10, IL-12 and VEGF in each supernatant were measured by ELISA assay. The number of Tca-8113 cells and the affect of supernatant on the lymphocyte conversion were subsequently determined by MTS assay.3. Human monocytes/macrophage system(THP-1 strains) was cultured under acid(p H 6.6 and p H 6.8) microenvironment and normal microenvironment(p H 7.2) containing different concentrations of TP for 24 h. The supernatants were collected after 24 h culture, and the content of VEGF and VEGF-C in each supernatant measured by ELISA assay. 4. Mononuclear cells separate from a healthy person’s peripheral blood were cultured under acid(p H 6.6, p H 6.8, p H 7.0) microenvironment and normal microenvironment(p H 7.2) with or without different concentrations of TP for 64 h. The expressions of CD68, CDl63 and CD80 on the surface of mononuclear macrophages were detected by flow cytometry. The contents of IL-10 and IL-12 in each supernatant were measured by ELISA assay. The affect of each supernatant on the lymphocyte conversion was subsequently determined by MTS. Tca-8113 cells were cultured under acid microenvironment and normal microenvironment containing different concentrations of TP with or without mononuclear cells for 64 h, and then the number of Tca-8113 cells was subsequently determined by MTS Assay.Results:1. With the decrease of p H value in cell culture fluid the number of Tca-8113 cells was increased and the content of IL-8, MCP-1 and HIF-1 in supernatant increased. 2. The number of CD68+ mononuclear macrophages did not change significantly in different culture condition. The number of M1 and M2 macrophages increased in acid-microenvironment. The content of IL-10 and VEGF were increased and the content of IL-12 was depressed in supernatant in acid-microenvironment. The lymphocyte conversion rate decreased when p H value descendent in cell culture fluid. The number of Tca-8113 cells increased when mixed with mononuclear macrophages.3. The content of VEGF and VEGF-C in THP-1 cells supernatant declined gradually with increasing TP concentration at the same p H value. 4. The number of CD80+ macrophages increased gradually with increasing TP concentration in acid microenvironment, however the number of CD163+ macrophages increased in the group of TP 10μg/ml and 25μg/ml and decreased in the in the group of TP 50μg/ml and 100μg/ml. The content of IL-10 was depressed and the content of IL-12 was increased in supernatant with increasing TP concentration in acid microenvironment. The lymphocyte conversion rate decreased with increasing TP concentration in acid microenvironment. Compared with the group of TP 0μg/ml, the number of Tca-8113 cells increased in TP(10μg/ml, 25μg/ml) and decreased in TP(50μg/ml,100μg/ml), but the number of Tca-8113 cells decreased in different TP concentration when mixed with mononuclear macrophages. Conclusion: Tca-8113 cells could change themselves to better adapt to the acidic microenvironment and unregulated multiple chemotactic factors which may caused infiltration and accumulation of mononuclear macrophages into tumor tissue. Acidic microenvironment can promote macrophage polarization, and present functional status of M2 macrophages, such as the expression of tumor growth factor was unregulated and the lymphocyte conversion rate down regulated. Low TP concentration could promote more monotype to M1 macrophage, and enhance the lymphocyte conversion rate, reduce the expression of tumor growth factor, thus produced antitumor function of mononuclear macrophages in the acidic microenvironment. The results obtained in this study indicate that the polarization and function of mononuclear macrophages can be changed after TP intervention in the acid microenvironment, but the mechanism need more research. |
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