| Objective:Melanoma is a skin neoplasm with high malignancy, which associated with immune dysfunction, while the role of cytokine in the host immune function has become an intensive consideration. IL-2, IFN-y and TNF-a with various biologic activities are important members of cytokines, which participate the immunoloregulation of the body and play important roles in resisting virus and tumor. Ganoderma lucidum polysaccharides (Gl-PS) is one of the main components of Ganoderma lucidum. Recently, many studies about the anti-tumor mechanism of Gl-PS have been reported. However, if it can antagonize the suppression on IL-2, IFN-y, TNF-a secretion by lymphocytes caused by supernatants of melanoma cells remains to be confirmed by straight evidence. In this study, subgroups were set up according to different concentration of Gl-PS in the wells with B16F10cell culture supernatant, and controled with RPMI1640perfect medium, to approach the antagonism of Ganoderma lucidum polysaccharides against suppression on IL-2, IFN-y, TNF-a secretion by lymphocytes caused by supernatants of melanoma cells.Methods:1. Cells:B16F10cell is a high metastastic melanoma cell line of C57BL/6j mouse origin.2. Experimental group and the interference:five groups have been set up in the experiment according to the concentrations of GL-PS in B16F10cell culture supernatant which culture the splenic mononuclear lymphocytes induced by PHA, controlled with RPMI-1640complete medium, namely,1640group (control group),0μg/ml group,0.2μg/ml group,0.8μg/ml group, 3.2μg/ml group,12.8μg/ml group.3. Immunocytochemical staining:the expression of IL-2, IFN-y, TNF-a in each group were detected by Immunocytochemical staining.4. Western blot:the expression of IL-2, IFN-y, TNF-a in each group were detected by Western blot as well.5. RT-PCR:the expression of IL-2, IFN-y, TNF-a mRNA in each group were detected by RT-PCR.6. The statistical analysis was performed with SPSS17.0using one-way analysis of variance (ANOVA). P<0.05was considered statistically significant.Result:1. Antagonism of Ganoderma lucidum polysaccharides against suppression caused by supernatants of melanoma cells on IL-2secretion in lymphocytes:Immunocytochemical staining test showed that, as compared with1640group, positive staining of IL-2was obviously weaker in supernatant group, and with the increasing of the Gl-PS concentration, positive staining of IL-2increased; Western blot test showed that the relative expression of IL-2in supernatant group was obviously weaker than in control group(P<0.05), and with the increasing of the Gl-PS concentration, the relative expression of IL-2increased, with a dose-dependent tendency. The analysis of variance indicated that difference was significant (F=10.326, P <0.01). Compared with0μg/ml group respectively by Gl-PS groups, the differences were all statistically significant (P<0.05); RT-PCR test showed that the IL-2mRNA expression in supernatant group was obviously weaker than in the control group (P<0.05), and with the increasing of the Gl-PS concentration, the expression of IL-2increased, which showed a dose-dependent tendency. The difference was significant according to analysis of variance (F=13.675, P<0.01). Compared with0μg/ml group respectively by Gl-PS groups, the differences were statistically significant in Gl-PS groups except0.2μg/ml group (P<0.05).2. Antagonism of Ganoderma lucidum polysaccharides against suppression caused by supernatants of melanoma cells on IFN-y secretion in lymphocytes:Immunocytochemical staining test showed that, as compared with1640group, positive staining of IFN-y was obviously weaker in supernatant group, and with the concentration of Gl-PS increasing, positive staining of IFN-γ increased; Western blot test showed that the expression of IFN-y in supernatant group was obviously weaker than in the control group (P <0.05), and with the increasing of the Gl-PS concentration, the expression of IFN-y increased, which showed a dose-dependent tendency. The analysis of variance indicated that the difference was significant (F=10.326, P<0.01). Compared with0μg/ml group respectively by Gl-PS groups, the difference was statistically significant in12.8μg/ml group (P<0.05); RT-PCR test showed that the IFN-y mRNA expression in supernatant group was obviously weaker than in the control group (P<0.05), and with the increasing of the Gl-PS concentration, the expression of IFN-y increased, which showed a dose-dependent tendency. The difference was significant according to analysis of variance (F=13.675, P<0.01). Compared with0μg/ml group respectively by Gl-PS groups, the differences were statistically significantin in Gl-PS groups except0.2μg/ml group (P<0.05).3. Antagonism of Ganoderma lucidum polysaccharides against suppression caused by supernatants of melanoma cells on TNF-a secretion in lymphocytes:Immunocytochemical staining test showed that, as compared with1640group, positive staining of TNF-a was obviously weaker in supernatant group, and with the increasing of the Gl-PS concentration, positive staining of TNF-a increased; Western blot test showed that the expression of TNF-a in supernatant group was obviously weaker than in the control group (p<0.05), and with the increasing of the Gl-PS concentration, the expression of TNF-a increased, which showed a dose-dependent tendency, while the analysis of variance indicated the difference was significant (F=5.143, P <0.01). Compared with Oμg/ml group respectively by Gl-PS groups, the differences were statistically significant in Gl-PS groups except0.2μg/ml group and0.8μg/ml group (P<0.05); RT-PCR test showed that the TNF-a mRNA expression in supernatant group was obviously weaker than in the control group (P<0.05), and with the increasing of the Gl-PS concentration, the relative expression of TNF-a increased, which showed a dose-dependent tendency. The difference was significant according to analysis of variance (F=8.143, P<0.01). Compared with0μg/ml group respectively by Gl-PS groups, the differences were statistically significant in groups except0.8μg/ml group (P<0.05).Conclusion:1. Suppression on IL-2, IFN-y, TNF-a secretion by lymphocytes can be caused by supernatants of melanoma cells2. Suppression on IL-2, IFN-y, TNF-a secretion by lymphocytes caused by supernatants of melanoma cells can be inhibited by Gl-PS. |
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