| Objective:Macrophages are immune cells of hematopoietic origin,which can provide vital innate immune defenses and have tissue-specific functions in the regulation and maintenance of organ homeostasis.Macrophages are an important part of innate immunity and a major participant in the mononuclear phagocyte system.They are effective antigen-presenting cells that can stably sense and respond to signals from the surrounding microenvironment,leading to immunogenicity or tolerance Sexual results.In the process of immune response,antigen information is transmitted to T lymphocytes by antigen presenting cells(APC),which activates T cells.T cell activation requires two kinds of signals.One is the interaction between TCR/CD3 complex and MHC molecule-antigen peptide complex to generate specific antigen stimulation signals for immune response;the other is costimulatory signals,such as CD80 and CD86.Regulate T lymphocyte proliferative response and induced effector function.Therefore,in order to exert the immune effect of macrophages,costimulatory molecules CD80,CD86 and MHC are indispensable.Ganoderma lucidum polysaccharides(Gl-PS)is one of the main biologically active components of Ganoderma lucidum,which has immunomodulatory,anti-angiogenic effects and potential anti-tumor activities.A large number of studies have shown that Ganoderma has a wide range of immunomodulatory effects,including promoting innate immune function,humoral immunity and cellular immunity.In particular,Gl-PS may affect immune cells,including B and T lymphocytes,dendritic cells,macrophages and natural killer cells,thereby promoting immune organ growth,cytokine release and other immune regulation functions.The anti-tumor effect of Gl-PS is closely related to its regulation of the immune function of the body.Therefore,it is still necessary to further study whether Gl-PS have an impact on macrophages and antigen-presenting molecules,and whether this effect is another way for Gl-PS to exert immune effect and anti-tumor effect.This experiment aims to explore the effects of Gl-PS on molecules related to antigen presentation of BALB/c mouse peritoneal macrophages,and to provide new ideas and theoretical basis for the treatment of infectious and malignant diseases of the immune system.Methods:1.Cell: The peritoneal macrophages used in this experiment were derived from BALB/c mice.2.Experimental grouping and intervention: In this experiment,Gl-PS was used to intervene mouse peritoneal macrophages for 48 h,and the groups were divided according to different concentrations,set 0ug/ml as the blank control group,set 0.2ug/ml,0.8ug/ml,3.2ug/ml,12.8ug/ml are four experimental groups.3.Flow cytometry: the expression of CD80,CD86,I-A/E in each group were detected by flow cytometry.4.RT-qPCR : the expression of CD80,CD86,I-A/E in each group were detected by RT-qPCR.5.Statistical analysis: SPSS 25.0 software was used for statistical analysis.Measurement data are expressed as(x±s).One-way analysis of variance(ANOVA)is used for comparison between multiple groups,and LSD-t is used for comparison between two groups.P<0.05 were considered to indicate a statistically significant difference.Results:1.The effect of Gl-PS on the costimulatory molecule CD80 of peritoneal macrophages in BALB/c mice.After treating peritoneal macrophages with Gl-PS(0.2,0.8,3.2,12.8ug/ml)for 48 hours,the positive expression rate of CD80 on the cell surface of each group was detected by flow cytometry,which were(0.38±0.33)%,(0.3±0.19)%,(2.06±1.30)%,(8.15±2.34)%,(17.74±5.40)%.It can be seen that with the continuous increase of the concentration of Gl-PS,the positive expression rate of CD80 in mouse peritoneal macrophages is getting higher and higher,showing a certain dose-dependent relationship.After one-way analysis of variance,the difference was statistically significant(P<0.05).The RT-qPCR technology showed that the expression levels of CD80 mRNA in mouse peritoneal macrophages were 1.00±0.00,4.79±3.15,7.46±3.14,17.99±10.71,63.23±8.94,and CD80 mRNA in the12.8ug/ml group could be seen the expression level of Gl-PS is the highest,so it can be concluded that with the continuous increase of the concentration of Gl-PS,the expression level of CD80 mRNA in mouse peritoneal macrophages increases successively,showing a certain dose-dependent relationship.According to the one-way analysis of variance,the differences Statistically significant(P<0.05).2.The effect of Gl-PS on the costimulatory molecule CD86 of peritoneal macrophages in BALB/c mice.After treating peritoneal macrophages with Gl-PS(0.2,0.8,3.2,12.8ug/ml)for 48 hours,the positive expression rate of CD86 on the cell surface of each group was detected by flow cytometry,which were(0.82±0.53)%,(0.96±0.74)%,(0.76±0.36)%,(26.60±12.29)%,(38.52±19.81)%.It can be seen that with the increasing concentration of Gl-PS,the positive expression rate of CD86 in mouse peritoneal macrophages is getting higher and higher,showing a certain dose-dependent relationship.After one-way analysis of variance,the difference was statistically significant(P<0.05).The RT-qPCR technique showed that the expression levels of CD86 mRNA in mouse peritoneal macrophages were1.00±0.00,3.93±2.34,12.20±11.73,45.01±1.64,186.11±74.25,and CD86 mRNA in the 12.8ug/ml group could be seen the expression level of Gl-PS is the highest,so it can be concluded that with the continuous increase of the concentration of Gl-PS,the expression level of CD86 mRNA in mouse peritoneal macrophages increases successively,showing a certain dose-dependent relationship.According to the one-way analysis of variance,the differences Statistically significant(P<0.05).3.The effect of Gl-PS on MHC class II molecules I-A/E of peritoneal macrophages in BALB/c mice.After treating the peritoneal macrophages with Gl-PS(0.2,0.8,3.2,12.8ug/ml)for 48 hours,the positive expression rates of IA/E on the cell surface of each group were detected by flow cytometry,respectively(22.46±6.28)%,(20.59±4.74)%,(39.33±11.50)%,(43.40±1.35)%,(51.39±15.27)%.It can be seen that with the increasing concentration of Gl-PS,the positive expression rate of I-A/E in mouse peritoneal macrophages is getting higher and higher,showing a certain dose-dependent relationship.After one-way analysis of variance,the difference was statistically significant(P<0.05).The RT-qPCR technology showed that the expression levels of IA/E mRNA in mouse peritoneal macrophages were1.00±0.00,7.17±7.84,15.88±10.80,48.61±17.32,344.96±301.19,and IA/E mRNA in the 12.8ug/ml group could be seen the expression level of Gl-PS is the highest,so it can be concluded that with the continuous increase of the concentration of Gl-PS,the expression level of IA/E mRNA in mouse peritoneal macrophages increases successively,showing a certain dose-dependent relationship.Factor analysis of variance showed that the difference was statistically significant(P<0.05).Conclusion:1.Ganoderma lucidum polysaccharides can up-regulate the costimulatory molecules CD80 and CD86 of mouse peritoneal macrophages.2.Ganoderma lucidum polysaccharides can up-regulate mouse peritoneal macrophages MHC class II molecules I-A/E. |
