| Objective: Taking Astragalus polysaccharide,Glycyrrhiza polysaccharide and their compound polysaccharides as research objects,using cell and molecular biology techniques,mouse regulatory T cells and human colon cancer HCT-116 cells are selected as target cells to study the effects of human colon cancer HCT-116 cells on mouse regulatory T cell differentiation and secretion of related cytokines from cell level,gene level and protein level.To study the effects of astragalus polysaccharide and licorice polysaccharide on differentiation of regulatory T cells and secretion of related cytokines in mice before and after co-culture with human colon cancer HCT-116 cells respectively.To study the effect of astragalus and licorice compound polysaccharide on the differentiation of regulatory T cells and secretion of related cytokines in co-cultured mice,and to compare the influence degree of astragalus polysaccharide,licorice polysaccharide and astragalus and licorice compound polysaccharide on regulatory T cells.The mechanism of their inducing regulatory T cell differentiation and anti-tumor activity was discussed in depth.Methods: 1.Flow cytometry was used to detect regulatory T cells in mice and determine the phenotype of the cells used in the experiment.2.CCK-8 method was used to detect the survival rate of regulatory T cells in mice after cultured for 24 hours with astragalus polysaccharide and licorice polysaccharide,and to determine the concentration range of drug without cytotoxicity.In the concentration range without cytotoxicity,the survival rate of regulatory T cells in mice after 72 hours of administration was detected,and the effect of administration time on the survival rate of regulatory T cells was excluded.The survival rate of HCT-116 cells after 72 hours of administration was detected,and the effect of drugs on the survival rate of HCT-116 cells in the co-culture model was excluded.3.Annexin V-FITC/PI double staining cell apoptosis detection kit was used todetect the apoptosis rate of regulatory T cells in mice 24 hours after administration,and to study the effect of drugs on the apoptosis rate.4.Transwell chamber established a non-contact co-culture model of HCT-116 cells and mouse regulatory T cells in vitro.Flow cytometry was used to detect the phenotypic changes of mouse regulatory T cells before and after co-culture.Real-time fluorescence quantitative PCR was used to detect the expression of mouse regulatory T cell related genes Foxp3,IL-10 and TGF-? m RNA before and after co-culture.Enzyme-linked immunosorbent assay was used to measure the secretion of IL-10 and TGF-? in mouse regulatory T cells before and after co-culture,so as to study the effect of HCT-116 cells on differentiation of mouse regulatory T cells and secretion of related cytokines.5.After cultured for 24 hours,the phenotypic changes of regulatory T cells in mice before and after co-culture were detected by flow cytometry.Three concentrations of Astragalus polysaccharide and Glycyrrhiza polysaccharide were selected.Annexin V-FITC/PI double staining cell apoptosis was used to detect the apoptosis rate of HCT-116 cells.Real-time fluorescence quantitative PCR was used to detect the expression of mouse regulatory T cell related genes Foxp3,IL-10 and TGF-? m RNA before and after co-culture.Enzyme-linked immunosorbent assay was used to measure the secretion of IL-10 and TGF-? in mouse regulatory T cells before and after co-culture,so as to study the effects of astragalus polysaccharide and glycyrrhiza polysaccharide on differentiation of mouse regulatory T cells and secretion of related cytokines before and after co-culture.6.CCK-8 method and Annexin V-FITC/PI double staining cell apoptosis method were used to detect the survival rate and apoptosis rate of mouse regulatory T cells and HCT-116 cells after cultured for 24 hours with 1: 1 astragalus polysaccharide and licorice polysaccharide of low,medium and high concentrations,excluding the effect of drugs on cell survival rate and apoptosis rate.7.The phenotypic changes of regulatory T cells in mice after co-culture with HCT-116 cells were detected by flow cytometry after administration of astragalus and licorice compound polysaccharide for 24 hours.Real-time fluorescence quantitative PCR was used to detect the expression of regulatory T cell related genes Foxp3,IL-10 and TGF-? m RNA in co-cultured mice.Enzyme-linked immunosorbent assay was used to measure the secretion of IL-10 and TGF-? in mouse regulatory T cells after co-culture,so as to study the effect of Astragalus and Glycyrrhiza complex polysaccharide on differentiation of regulatory T cells and secretion of relatedcytokines in mice after co-culture.Results: 1.The phenotype of mouse regulatory T cells used in this experiment is CD4+CD25+Foxp3+CD152+PD1-/+.2.According to CCK-8 method,the cytotoxic concentration of Astragalus polysaccharide and Glycyrrhiza polysaccharide on regulatory T cells in mice is0-250?g/m L.The final concentration of 0-50?g/m L drug was selected.The results showed that 72 h after drug culture had no effect on the survival rate of mouse regulatory T cells and HCT-116 cells.3.Annexin V-FITC/PI double staining cell apoptosis detection results show that astragalus polysaccharide and licorice polysaccharide with final concentration of0-50?g/m L have no effect on apoptosis rate of regulatory T cells in mice after 24 hours of administration.4.The results of flow cytometry show that HCT-116 cells can enhance the expression of mouse regulatory T cell phenotype after co-culture,with CD25 shift being the most obvious,followed by Foxp3,CD4,PD-1,and CD152;Real-time fluorescence quantitative PCR showed that human colorectal cancer HCT-116 cells up-regulated the expressions of Foxp3,IL-10 and TGF-? m RNA in mouse regulatory t cells,of which IL-10 m RNA expression was significantly increased(P<0.01)and TGF-?m RNA expression was increased(P<0.05).Enzyme-linked immunosorbent assay showed that HCT-116 cells of human colorectal cancer increased the secretion of IL-10 and TGF-? in regulatory T cells of mice,with significant difference(P<0.01).5.After 24 hours of culture with the final concentration of 0-50?g/m L of Astragalus polysaccharide and Glycyrrhiza polysaccharide,the flow cytometric measurement showed that the expression of CD4 on the surface of regulatory T cells in mice was significantly reduced,followed by CD25,which was concentration dependent.For phenotypes Foxp3,PD-1 and CD125,there is a tendency to decrease the expression,but none is obvious.Therefore,the final concentrations of drugs selected in the follow-up experiments are: 10,25,50 ?g/ml astragalus polysaccharide and licorice polysaccharide.The administration time is 24 h,and CD4 and CD25;are selected as indexes.Real-time fluorescence quantitative PCR further determined the regulation of astragalus polysaccharide and licorice polysaccharide on gene level.The results showed that the expressions of Foxp3,IL-10,TGF-? m RNA in regulatory T cells of mice before and after co-culture after drug treatment were down-regulated aswell as those in the untreated group.At the concentration of 50?g/m L,the down-regulation of Foxp3,IL-10,TGF-? m RNA was significantly different from that of the control group(P<0.05).Enzyme-linked immunosorbent assay showed that the drug reduced the secretion of IL-10 and TGF-? in regulatory T cells of mice before and after co-culture.6.The results of CCK-8 and Annexin V-FITC/PI double staining cell apoptosis method showed that the survival rate and apoptosis rate of mouse regulatory T cells and HCT-116 cells were not affected after 24 hours of culture with the final concentration of 10,25,50 ?g/ml of Astragalus and Glycyrrhiza complex polysaccharide(1: 1).7.From the point of view of protein level and gene level,through flow cytometry,real-time fluorescence quantitative PCR and enzyme-linked immunosorbent assay,it is found that the effect of Astragalus and Glycyrrhiza complex polysaccharide on regulatory T cells is better than that of Astragalus and Glycyrrhiza polysaccharide,which is manifested in down-regulating the expression of CD4 and CD25,reducing the expression of Foxp3,IL-10 and TGF-? m RNA,and down-regulating the secretion of IL-10 and TGF-?.Conclusion: 1.HCT-116 cells can promote the differentiation of regulatory T cells in mice,up-regulate the expression of immunosuppressive molecules on the cell surface,and make cells differentiate into immunosuppressive cells.2.Through co-culture,it is found that Astragalus polysaccharide and Glycyrrhiza polysaccharide inhibit tumor immune escape and participate in tumor immune regulation by inducing differentiation of regulatory T cells,down-regulating the expression of immunosuppressive molecules and related m RNA,and antagonizing the effect of human colon cancer cells on mouse regulatory T cells.3.Astragalus polysaccharide and Glycyrrhiza polysaccharide have no killing effect on tumor cells.They participate in tumor immunity by inhibiting the cellular function of regulatory T cells in mice.4.Astragalus and Glycyrrhiza compound polysaccharide induces the differentiation of regulatory T cells in mice to immunosuppression direction,and is superior to Astragalus and Glycyrrhiza polysaccharide in down-regulating the expression of Foxp3,IL-10 and TGF-? m RNA and reducing the secretion of IL-10 and TGF-?.To sum up,APS and GP are likely to participate in the immune regulation and anti-tumor effects in vivo and in vitro by down-regulating the expression of Tregs immunosuppressive molecules,and the effect of combined medication is better than that of single medication,which provides a strong basis for the clinical rational use ofAstragalus polysaccharide and Glycyrrhiza polysaccharide. |
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