| Objective:For a long time, macrophages has been considered a very important immune cells.Macrophages express a variety of antigen presenting related surface molecules, which can intake antigen by phagocytosis, pinocytosis role and the function of the receptor mediated cell swallowing.Which can also express a large number of MHCI and MHCII molecules and stimulating molecules such as CD80, CD86, CD40, processing antigen in cell. So, macrophages have strong antigen presenting ability and the consuming digest function.macrophages can secrete a large number of promoting inflammatory cvtokines such as IL-12, IL-23, IL-1, IL-6, IL-13, TNF and chemokines CCL-2, CCL-3 etc, activation of Thl immune response, secretion by high levels of cytotoxic intermediates such as NO, reactive oxygen intermediates mediated killing tumor cells and digestion of microbes in the cell. Therefore macrophages are important effect cells of the body’s immune defense and anti-tumor.macrophages can also secrete high level of immune inhibitory cytokines such as IL-10, TGF-β and VEGF, suppress inflammation and immune response. recently years, many studies have shown that macrophages are the main composition of white blood cells infiltrating into tumor tissue.In the early days of the tumor, macrophage give play to immune surveillance and antitumor effect by killing tumor cells and presenting tumor related antigen, in the late days of the tumor, macrophages can release many cytokines and chemokines, promoting angiogenesis and tumor growth, and can promote the tumor invasion and metastasis by the degradation of matrix. Therefore, understanding the role of macrophages in tumor, adjusting the function of macrophages and macrophage as important targets for tumor immunotherapy, make its immune surveillance and antitumor effect.The ganoderma lucidum polysaccharide (GL-PS) is one of the important medicinal components of ganoderma lucidum, which have the effect of immune enhancement and antitumor.Currently, research on the antitumor mechanism of ganoderma lucidum polysaccharides more concentrated on the immune function and influence on the telomerase activity and differentiation, etc.By the research, ganoderma lucidum polysaccharide have not direct tumor suppression and the role of inducing tumor cell apoptosis in vitro and its anti-tumor mechanism could be adjust host immune function, enhance immune cell activity, thereby inhibiting tumor development. Study confirms, ganoderma lucidum polysaccharide can activate T cells in vivo and in vitro, and enhance the phagocytosis of macrophage, and promote the NO, IL-1β, TNF-a production and release, regulate the body’s immune function.Zhibin Lin proved that ganoderma lucidum polysaccharide having no direct anti-tumor effect by serum pharmacology experiment as well as a variety of in vivo and in vitro test method, its antitumor effect is by promoting TNF-a, IFN-y mRNA expression, increasing production of TNF-a, IFN-y, thereby inhibiting tumor cells and promoting its apoptosis. Thus, the antitumor function of the ganoderma lucidum polysaccharide is closely related to the reg ulate of the body’s immune function.the ganoderma lucidum polysaccharides impact will be on macrophages and their secretion of cytokines, will be what kind of impact, whether the effect of ganoderma lucidum polysaccharide antitumor another way.This study aims to reveal the new anti-tumor mechanism of Gl-PS by discussing the effections of G1-PS on TNF-α, CCL-2, IL-6, IL-10 and IL-12 of mice peritoneal macrophages activitied by LPS.Methods:1. Cells:Mice Peritoneal Macrophages from C57BL/6 j mice.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 Mice Peritoneal Macrophages, namely 0μg/ml group 0.2μg/ml group,0.8μg/ml group,3.2μg/ml group,12.8μg/ml group, set up 0μg/ml group as a control group.Each group contains the same concentration of Lipopolysaccharides (LPS) 10μg/ml.3. ELISA:the expressions of TNF-α, CCL-2, IL-6, IL-10 and IL-12 in each group were detected by ELISA.4. q-PCR:the expression of TNF-a, CCL-2, IL-6, IL-10 and IL-12 in each group were detected by q-PCR.5. The statistical analysis was performed with SPSS17.0 using one-way analysis of variance (ANOVA). P<0.05 was considered statistically significant.Result:1.The effect of G1-PS on TNF-a secretion in Mice Peritoneal Macrophages with the induction by LPS was detected at the cytokines secretion 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 Mice Peritoneal Macrophages with the induction by LPS cultured for 24h.The culture supernatant level of TNF-α (pg/ml) was detected by ELISA were 310.705±18.196,378.631±15.448,379.777±20.712,391.189±24.264, 403.730±29.753, the assays showed that with the concentration of GL-PS increasing, the level of TNF-α was getting higher and higher, rendering a dose-dependent relationship.The analysis of variance indicated the difference was significant (F=11.692, P<0.01). q-PCR test showed that the TNF-α mRNA expression in each group of Mice Peritoneal Macrophages were 1.000±0.000,1.081±0.003,1.083±0.003,1.085±0.005,1.087±0.002. And the 12.8μg/ml group also high express TNF-a. With the concentration of G1-PS increasing, the TNF-a mRNA expression rising, which showed a dose-dependent tendency. The difference was significant by analysis of variance (F=506.873, P<0.01)2.The effect of G1-PS on CCL-2 secretion in Mice Peritoneal Macrophages with the induction by LPS was detected at the cytokines secretion and gene level:under the action of different concentrations of Gl-PS (0μg/ml,0.2μg/ml,0.8μg/ml,3.2μg/ml,12.8μg/ml), each group of Mice Peritoneal Macrophages with the induction by LPS cultured for 24h.The culture supernatant level of CCL-2 (pg/ml) was detected by ELISA were 26.827±8.660,84.235±7.722,93.929±6.937,98.053±20.500,97.570±13.505, the assays showed that with the concentration of GL-PS increasing, the level of CCL-2 was getting higher and higher, rendering a dose-dependent relationship.The analysis of variance indicated the difference was significant (F=35.084, P<0.01). q-PCR test showed that the CCL-2 mRNA expression in each group of Mice Peritoneal Macrophages were 1.000±0.000, 1.055±0.004,1.057±0.006,1.059±0.004,1.060±0.002. And the 12.8μg/ml group also high express CCL-2. With the concentration of G1-PS increasing, the CCL-2 mRNA expression rising, which showed a dose-dependent tendency. The difference was significant by analysis of variance (F=170.489, P<0.01)3. The effect of G1-PS on IL-6 secretion in Mice Peritoneal Macrophages with the induction by LPS was detected at the cytokines secretion and gene level:under the action of different concentrations of G1-PS (0μg/ml, 0.2 μg/ml,0.8 μg/ml,3.2μg/ml,12.8 μg/ml), each group o f Mice Peritoneal Macrophages with the induction by LPS cultured for 24h.The culture supernatant level of IL-6 (pg/ml) was detected by ELISA were 339.236±29.488,404.431±36.023,395.986±13.884,423.251±19.502 450.093±38.936, the assays showed that with the concentration of GL-PS increasing, the level of IL-6 was getting higher and higher, rendering a dose-dependent relationship.The analysis of variance indicated the difference was significant (F=15.375, P<0.01). q-PCR test showed that the IL-6 mRNA expression in each group of Mice Peritoneal Macrophages were 1.000±0.000, 1.031±0.003,1.033±0.004,1.039±0.004,1.048±0.012. And the 12.8μg/ml group also high express IL-6. With the concentration of G1-PS increasing, the IL-6 mRNA expression rising, which showed a dose-dependent tendency. The difference was significant by analysis of variance (F=27.382, P<0.01)4.The effect of G1-PS on IL-10 secretion in Mice Peritoneal Macrophages with the induction by LPS was detected at the cytokines secretion 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 Mice Peritoneal Macrophages with the induction by LPS cultured for 24h.The culture supernatant level of IL-10 (pg/ml) was detected by ELISA were 102.901±18.713,88.399±6.557,86.397±11.235,73.887±5.202, 59.385± 13.337, the assays showed that with the concentration of GL-PS increasing, the level of IL-10 was getting lower and lower, rendering a dose-dependent relationship.The analysis of variance indicated the difference was significant (F= 11.123, P<0.01). q-PCR test showed that the IL-10 mRNA expression in each group of Mice Peritoneal Macrophages were 1.000±0.000, 0.987±0.006,0.982±0.002,0.981±0.002,0.974±0.004. And the Oμg/ml group also high express IL-10. With the concentration of Gl-PS increasing, the IL-10 mRNA expression reduced, which showed a dose-dependent tendency. The difference was significant by analysis of variance (F=27.094, P<0.01)5.The effect of GI-PS on IL-12 secretion in Mice Peritoneal Macrophages with the induction by LPS was detected at the cytokines secretion and gene level:under the action of different concentrations of G1-PS (Oμg/ml, 0.2μg/ml,0.8μg/ml,3.2μg/ml,12.8μg/ml), each group of Mice Peritoneal Macrophages with the induction by LPS cultured for 24h.The culture supernatant level of IL-12 (pg/ml) was detected by ELISA were143.919±8.320,165.085±10.533,178.367±20.209,189.783±22.350, 206.916±5.059, the assays showed that with the concentration of GL-PS increasing, the level of IL-12 was getting higher and higher, rendering a dose-dependent relationship.The analysis of variance indicated the difference was significant (F=15.464, P<0.01). q-PCR test showed that the IL-12 mRNA expression in each group of Mice Peritoneal Macrophages were 1.000±0.000,1.014±0.005,1.019±0.002,1.023±0.006,1.026±0.002. And the 12.8μg/ml group also high express IL-12. With the concentration of G1-PS increasing, the IL-12 mRNA expression rising, which showed a dose-dependent tendency. The difference was significant by analysis of variance (F=26.436, P<0.01)Conclusion:1.The ability that Mice Peritoneal Macrophages secrete cytokines TNF-a, CCL-2, IL-6, IL-12 can be increased by Ganoderma lucidum polysaccharides.2.The ability that Mice Peritoneal Macrophages secrete cytokines IL-10 can be reduced by Ganoderma lucidum polysaccharides. |
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