| Objective:Macrophages derived from bone marrow CD34+ progenitor cells and the latter one have proliferated and been in the form of monocytes releaseing into the blood. And then seeping to the organization, they develop into Mature and specific macrophages. Macrophages have high heterogeneity. Different stimulus and some microorganism, etc all can stimulate macrophage activatiing different phenotypic and functional characteristic subtypes, which is called macrophages heterogeneity. The heterogeneity depend on the undifferentiated monocytes heterogeneity in organization and microenvironment. On the basis of phenotypic, functional characteristic and the immune response to the Th1/Th2, They are divided into M1 and M2. M1 classically activated macrophages, It can kill bacteria, secrete a variety of inflammatory factors, present antigen and start adaptive immune response.M2, alternatively activated macrophages. It can be divided into three subsets depend on different activation marks, which have strong functions of immunomodulatory effects and wound healing.We focus on the correlation of macrophages heterogeneity with diseaes, from this we explore the mechanism and treatment of tumor. For example in the pathological state inducing beneficial macrophage subsets and suppressing harmful macrophage subsets and characteristics, targeted treatment for tumor. In the tumor tissue, M1, M2 macrophages are co-existence. In recent decades, the study reveal that there are a large number of macrophages in the tumor stroma, named tumor associated macrophages(TAM). In recent years, the study reveal that TAM is similar to M2 on the phenotypic characteristics, which does not play a role of anti-tumor on the function, but to be involved in tumorigenesis development, invasion and metastasis. So M2 macrophages has become a potential new targets for cancer treatment.Ganoderma lucidum has more than 2000 years in China’s history and the ganoderma lucidum polysaccharide is one of the main efficacy components. It has extensive pharmacological activities, such as improving the body’s immune function, anti-tumor, etc. The current research about ganoderma lucidum polysaccharide anti-tumor mechanism has been reported a lot. But by adjusting M2 macrophages to secrete cytokines to achieve anti-tumor effect has not been reported too much. This experiment throueh observing the level of cytokine and genes of M2 macrophages variation to different concentration of ganoderma lucidum polysaccharides, then we explore the ganoderma lucidum polysaccharide by adjusting the peritoneal macrophages activity to play a role of anti-tumor, which provide the basis for clinical application.Methods:1.Cells:The cells in this experiment derive from C57BL/6 mice peritoneal macrophage.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 peritoneal macrophage, Oμg/ml group (control group), 0.2μg/ml group,0.8μg/ml group,3.2μg/ml group,12.8μg/ml group.3.ELISA:detection the expression of IL-12、IL-10、IL-6、MCP-1 and TNF-a in every groups.4.Real-time-PCR:detection the expression of IL-12、IL-10、IL-6、 MCP-1 and TNF-a in every groups.5. Statistical analysis:Through SPSS17.0 statistical software.Comparison between groups use one-way analysis of variance (ANOVA) and Dunnett-t. P<0.05 was considered statistically significant.Result:1. From the secretion of cytokines and gene level the impact on IL-10 secreted by ganoderma lucidum polysaccharides on mouse peritoneal macrophages induced by IL-4:Extracting in mouse peritoneal macrophages after overnight and then cultured under the different concentrations of GL-PS (0μg/ml、0.2μg/ml、0.8μg/ml、3.2μg/ml、12.8μg/ml)) and the inducer IL-4 for 24h. It was detected by ELISA. The expression of IL-10 (pg/ml) is 327.187±21.050、305.077±14.610、291.376±18.410、274.543±15.312、 255.427±19.930. With increasing concentration of ganoderma lucidum polysaccharide, the expression of IL-10 get lower and lower, presenting a certain dose-response relationship. After analysis of variance, they have significant difference (F=7.015, P<0.05). It was detected by Real-time PCR. The expression of IL-10 mRNA is 1.000±0.000、0.921±0.095、0.557±0.040、 0.460±0.030、0.354±0.017.0.2ug/ml express relatively high. With increasing concentration of ganoderma lucidum polysaccharide, the expression of IL-10 get lower and lower, presenting a certain dose-response relationship. After analysis of variance, they have significant difference (F=105.708, P<0.05)2. From the secretion of cytokines and gene level the impact on IL-12 secreted by ganoderma lucidum polysaccharides on mouse peritoneal macrophages induced by IL-4:Extracting in mouse peritoneal macrophages after overnight and then cultured under the different concentrations of GL-PS (0μg/ml、0.2μg/ml、0.8μg/ml、3.2μg/ml、12.8μg/ml) and the inducer IL-4 for 24h. It was detected by ELISA. The expression of IL-12 (pg/ml) is 59.677±12.828、72.833±11.743、90.837±12.221、103.223±14.318 113.200±11.954.With increasing concentration of ganoderma lucidum polysaccharide, the expression of IL-12 get higher and higher, presenting a certain dose-response relationship. After analysis of variance, they have significant difference (F=9.000,P<0.05). It was detected by Real-time PCR.The expression of IL-12 mRNA is 1.000±0.000、1.007±0.020、 1.087±0.035、1.115±0.027、1.609±0.043.12.8ug/ml express relatively high. With increasing concentration of ganoderma lucidum polysaccharide, the expression of IL-12 get higher and higher, presenting a certain dose-response relationship.After analysis of variance, they have significant difference (F=497.089, P<0.05)3. From the secretion of cytokines and gene level the impact on IL-6 secreted by ganoderma lucidum polysaccharides on mouse peritoneal macrophages induced by IL-4:Extracting in mouse peritoneal macrophages after overnight and then cultured under the different concentrations of GL-PS (0μg/ml、0.2(ig/ml、0.8μg/ml、3.2μg/ml、12.8μg/ml) and the inducer IL-4 for 24h. It was detected by ELISA.The expression of IL-6 (pg/ml) is 319.103±23.928、330.117±14.915.355.163±26.263、387.690±22.752、 416.700±27.568. With increasing concentration of ganoderma lucidum polysaccharide, the expression of IL-6 get higher and higher, presenting a certain dose-response relationship. After analysis of variance, they have significant difference (F=8.899, P<0.05). It was detected by Real-time PCR. The expression of IL-6 mRNA is 1.000±0.000、1.141±0.045、1.195±0.028、 1.358±0.029、1.439±0.026.12.8ug/ml express relatively high. With increasing concentration of ganoderma lucidum polysaccharide, the expression of IL-6 get higher and higher, presenting a certain dose-response relationship.After analysis of variance, they have significant difference (F=128.101, P<0.05)4. From the secretion of cytokines and gene level the impact on TNF-a secreted by ganoderma lucidum polysaccharides on mouse peritoneal macrophages induced by IL-4:Extracting in mouse peritoneal macrophages after overnight and then cultured under the different concentrations of GL-PS (Oμg/ml、0.2μg/ml、0.8μg/ml、3.2μg/ml、12.8μg/ml) and the inducer IL-4 for 24h. It was detected by ELISA.The expression of TNF-α (pg/ml) is 25.860±4.798、31.750±5.685、43.753±6.617、54.137±7.341、62.030±7.65. With increasing concentration of ganoderma lucidum polysaccharide, the expression of TNF-a get higher and higher, presenting a certain dose-response relationship. After analysis of variance, they have significant difference (F=16.053, P<0.05). It was detected by Real-time PCR. The expression of TNF-a mRNA is 1.000±0.000、1.071±0.057、1.176±0.037、1.258±0.023、 1.446±0.072.12.8ug/ml express relatively high. With increasing concentration of ganoderma lucidum polysaccharide, the expression of TNF-a get higher and higher, presenting a certain dose-response relationship. After analysis of variance, they have significant difference (F=43.750,P<0.05)5. From the secretion of cytokines and gene level the impact on MCP-1 secreted by ganoderma lucidum polysaccharides on mouse peritoneal macrophages induced by IL-4:Extracting in mouse peritoneal macrophages after overnight and then cultured under the different concentrations of GL-PS (0μg/ml、0.2μg/ml、0.8μg/ml、3.2μg/ml、12.8μg/ml) and the inducer IL-4 for 24h. It was detected by ELISA.The expression of MCP-1 (pg/ml) is 25.913±7.113、37.617±6.541、49.797±8.879、80.790±7.174、90.967±7.266. With increasing concentration of ganoderma lucidum polysaccharide, the expression of MCP-1get higher and higher, presenting a certain dose-response relationship. After analysis of variance, they have significant difference (F=42.230, P<0.05). It was detected by Real-time PCR. The expression of MCP-1 mRNA is 1.000±0.000、1.030±0.016、1.122±0.027、1.136±0.023、 1.215±0.026.12.8ug/ml express relatively high. With increasing concentration of ganoderma lucidum polysaccharide, the expression of MCP-1 get higher and higher, presenting a certain dose-response relationship. After analysis of variance, they have significant difference (F=143.112, P<0.05)Conclusion:1. Mice peritoneal macrophages induced by IL-4 can express high IL-10, low IL-12、IL-6、TNF-α、MCP-1.2. Ganoderma lucidum polysaccharide can promote the secretion of I cytokines IL-12、IL-6、TNF-α、MCP-1 and inhibit the secretion of II cytokines IL-10. The characteristics of M2 macrophages has been weaken, so as to weaken its feature of promoting tumor. |
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