| Objective:Melanoma is a malignant tumor derived from melanocytes, occurs mostly in the patients over the age of 40, more common in the skin. Although it appears first on the surface of the body in most patients, which can be discovered early and then controlled by surgery,1/3 of patients recur or distantly metastasize, leading to insensitivity for radiotherapy and chemotherapy, more progression, and poor prognosis, which seriously threats human health. Besides surgery, chemotherapy and radiotherapy, the immunotherapy has become an intensive consideration.Tumor immunology research revealed that the generation and development of tumor is closely related not only to the immune function, but also to immunosuppressive action induced by tumor cells in the host. The tumor cells secrete immunosuppressive substances to inhibit immune function, which is the important mechanism of tumor escaping immunosurveillance and plays an important role in promoting tumor generation and development.Ganoderma lucidum has 2000 years of medical history in China, and Ganoderma lucidum polysaccharides (G1-PS) is one of its main components. Gl-PS has multiple pharmacologic actions including improvement of immune function and anti-tumor. Recently, many studies about the anti-tumor mechanism of Gl-PS have been reported in the literature. However, the domestic and abroad reports about the effect of Gl-PS on immunosuppressive molecules secretion in tumor cells are still rare. B16F10 cells are highly metastatic melanoma cells derived from C57BL/6j mice. It has been certified in experiment that TGF-β1, IL-10 and VEGF can be detected in the supernatant of B16F10 cells culture.To reveal the new anti-tumor mechanism of Gl-PS, and provide new ideas for the immunotherapy of melanoma, the effect of Ganoderma lucidum polysaccharides on immunosuppressive molecules secretion in B16F10 cells was explored in this study.Methods:1. Cells:B16F10 cell is a high metastastic melanoma cell line of C57BL/6j mouse origin.2. Experimental group and the intervention:five groups have been set up in the experiment according to the concentrations of GL-PS in RPMI-1640 complete medium of B16F10 cells, namely 0μg/ml group (control group), 0.2μg/ml group,0.8μg/ml group,3.2μg/ml group,12.8μg/ml group.3. Immunocytochemical staining:the expression of VEGF, TGF-β1, IL-10 in each group were detected by Immunocytochemical staining.4. Western blot:the expression of VEGF, TGF-β1, IL-10 in each group were detected by Western blot.5. PT-PCR:the expression of VEGF, TGF-β1, IL-10 in each group were detected by PT-PCR.6. The statistical analysis was performed with SPSS 17.0 using one-way analysis of variance (ANOVA). P<0.05 was considered statistically significant.Result:1. The effect of G1-PS on VEGF secretion in B16F10 cells was detected at the protein and gene level:under the action of different concentrations of G1-PS (0μg/ml,0.2μg/ml,0.8μg/ml,3.2μg/ml,12.8μg/ml), each group of B16F10 cells cultured for 48h, then detected by immunocytochemical staining, and positive staining was mainly localized in the cytoplasm, membrane and nucleus. The high expression were found in the control group and 0.2μg/ml group. With the concentration of G1-PS increasing, VEGF positive staining becomes weaker and weaker; Western blot test showed that the relative expression of VEGF in each group of B16F10 cells were 1.628±0.467,1.769 ±0.159,0.742±0.066,0.825±0.069,0.414±0.103. The control group and 0.2μg/ml group still high expressed VEGF, and with the concentration of G1-PS increasing, the relative expression of VEGF decreased, which showed a dose-dependent tendency. The analysis of variance indicated the difference was significant (F=19.917, p<0.01). Mutiple comparisons, except for 0μg/ml group vs 0.2μg/ml group,0.8μg/ml group vs 3.2μg/ml group,3.2μg/ml group vs 12.8μg/ml group,0.8μg/ml group vs 12.8μg/ml group, the remaining differences among the groups were statistically significant (p<0.05); RT-PCR test showed that the VEGF mRNA expression in each group of B16F10 cells were 1.283±0.159、1.130±0.087、0.970±0.079、0.880±0.061、0.750±0.140. And the control group also high express VEGF. With the concentration of G1-PS increasing, the VEGF mRNA expression decreased, which showed a dose-dependent tendency. The difference was significant by analysis of variance (F=10.508, p<0.01). Mutiple comparisons, the differences were statistically significant (p<0.05) between 0μg/ml group vs 0.8μg/ml group, 0μg/ml group vs 3.2μg/ml group,0μg/ml group vs 12.8μg/ml group,0.2μg/ml group vs 12.8μg/ml group. These results suggest that VEGF gene transcription in B16F10 cells can be inhibited by G1-PS.2. The effect of G1-PS on TGF-β1 secretion in B16F10 cells was detected at the protein and gene level:under the action of different concentrations of G1-PS (0μg/ml,0.2μg/ml,0.8μg/ml,3.2μg/ml,12.8μg/ml), each group of B16F10 cells cultured for 48h, then detected by immunocytochemical staining, and positive staining was mainly localized in the cytoplasm, nucleus and nuclear membrane. The high expression were found in the control group. With the concentration of G1-PS increasing, TGF-β1 positive staining becomes weaker and weaker; Western blot test showed that the relative expression of TGF-β1 in each group of B16F10 cells were1.949±0.153、1.841±0.179、0.770±0.297、0.627±0.284、0.314±0.268.The control group still high expressed TGF-β1, and with the concentration of G1-PS increasing, the relative expression of TGF-β1 decreased, which showed a dose-dependent tendency. The analysis of variance indicated the difference was significant (F=6.027, p<0.05). Mutiple comparisons, the differences were statistically significant (p<0.05) between 0μg/ml group vs 12.8μg/ml group, 0.2μg/ml group vs 12.8μg/ml group; RT-PCR test showed that the TGF-β1 mRNA expression in each group of B16F10 cells were 1.277±0.117、1.190±0.087、1.023±0.049、0.827±0.076、0.697±0.112. The expression of TGF-β1 in the control group was highest. With the concentration of G1-PS increasing, the TGF-β1 mRNA expression decreased, which showed a dose-dependent tendency. The analysis of variance indicated the difference was significant (F=20.934,p<0.01). Mutiple comparisons, except for 0μg/ml group vs 0.2μg/ml group,0.2μg/ml group vs 0.8μg/ml group,0.8μg/ml group vs 3.2μg/ml group,3.2μg/ml group vs 12.8μg/ml group, the remaining differences among the groups were statistically significant (p<0.05). These results suggest that TGF-β1 gene transcription in B16F10 cells can be inhibited by G1-PS.3. The effect of G1-PS on IL-10 secretion in B16F10 cells was detected at the protein and gene level:under the action of different concentrations of G1-PS (0μg/ml,0.2μg/ml,0.8μg/ml,3.2μg/ml,12.8μg/ml), each group of B16F10 cells cultured for 48h, then detected by immunocytochemical staining, and positive staining was mainly localized in the the cytoplasm. The high expression were found in the control group. With the concentration of G1-PS increasing, IL-10 positive staining becomes weaker and weaker; Western blot test showed that the relative expression of IL-10 in each group of B16F10 cells were 1.477±0.439、1.589±0.317、0.934±0.129、0.892±0.268、 0.400±0.292. The relative expression of IL-10 was higher in control group and 0.2μg/ml group. With the concentration of G1-PS increasing, the relative expression of IL-10 decreased, which showed a dose-dependent tendency. The analysis of variance indicated the difference was significant (F=7.498,p<0.01). Mutiple comparisons, the differences were statistically significant (p<0.05) between 0μg/ml group vs 12.8μg/ml group,0.2μg/ml group vs 12.8μg/ml group; RT-PCR test showed that the IL-10 mRNA expression in each group of B16F10 cells were 1.303±0.160、1.157±0.025、0.923±0.012、0.863±0.064、 0.750±0.123. And the expression of IL-10 was highest in control group. With the concentration of G1-PS increasing, the IL-10 mRNA expression decreased, which showed a dose-dependent tendency. The difference was significant by analysis of variance (F=16.806,p<0.01). Mutiple comparisons, except for 0μg/ml group vs 0.2μg/ml group,0.2μg/ml group vs 0.8μg/ml group,0.8μg/ml group vs 3.2μg/ml group,3.2μg/ml group vs 12.8μg/ml group,0.8μg/ml group vs 12.8μg/ml group, the remaining differences among the groups were statistically significant (p<0.05). These results suggest that IL-10 gene transcription in B16F10 cells can be inhibited by G1-PS.Conclusion:1. B16F10 melanoma cells can secrete immunosuppressive molecules VEGF,TGF-β1, IL-10.2. The ability that B16F10 cells secrete immunosuppressive molecules VEGF,TGF-β1, IL-10 can be reduced by Ganoderma lucidum polysaccharides. |
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