| Polysaccharides have a variety of biological activity,such as enhancing the body's immune function,anti-aging,anti-cancer,blood sugar and so on.Molecular modification of polysaccharides to change the polysaccharide space structure,the type of substituents,the number and location can enhance its activity or increase the new biological activity.After structural modification,the immune effect many polysaccharides were significantly improved,such as sulfated Ophiopogon japonicus polysaccharide,carboxymethylated astragalus polysaccharide,selenium angelica polysaccharide and so on.Some polysaccharides modified by hydroxyethylation,the immune activity was significantly improved,such as Poria polysaccharides,Achyranthes bacillus and so on.A large number of studies have shown that Hericium erinaceus polysaccharides can improve the immunity of the body,and the previous study shows that hydroxyethylation can improve the activity of Hericium erinaceus polysaccharides on DCs antigen activity and promote IL-12,IL-18 and IFN-? and other imnune factors,and because the interaction of DC cells and NK cells in the body has play an important role in immunization,so this test is aimd to modificate the Hericium polysaccharide by hydroxyethylation,and to explore the hydroxy effects of Polysaccharides from Hericium erinaceus on the interaction of DC Cells and NK Cells.The experiment is divided into the following sections:1.The polysaccharide was modified by hydroxyethylation to obtain HE-HEP.2.The establishment of cell model:Bone marrow cells were extracted from the femur and tibia of mice,and rmIL-4 and rmGM-CSF were used to induce dendritic cells to mature in vitro.Splenic lymphocytes were extracted from the spleen of mice cells,and constantly purifing mice to obtain natural spleen cells by rmIL-2.The morphological changes of the cells were observed under an optical microscope and the expression of CD40,CD86,CD11c and MHC ? and NK cells CD 107a and CD49b were detected by flow cytometry.Two kinds of cells were mixed and cultured in 1:5 ratio to establish DC cell and NK cell interaction model.3.Firstly,the safe concentration of HEP and HE-HEP was obtained by polysaccharide virulence test,then the polysaccharide was applied to DC cells,and DC cells were applied to T lymphocytes to obtain the optimal concentration of polysaccharides in T lymphocytes when DC cells were added.Then acting the HE-HEP and HEP on both cells,the levels of IL-12,IL-15 and IL-18 were detected by ELISA.The expression of IL-12,IL-15,IL-18,NF-?B,GM-CSF,IFN-?,TNF-? and NKG2D mRNA were detected by RT-PCR.4.The levels of I?B-?,I?B-? and NF-?B p65 and NF-?B p50 in the cytoplasmic protein were detected by western blot.The results showed that:(1)NK cells could enhance the ability of antigenization of mature DC cells.DC cells were cultured alone or NK cells and DC cells mixed with 1:5 ratio,a certain concentration of HEP and HE-HEP can enhance the ability of antigenization of mature DC cells.(2)NK cells and DC cells mixed culture can make IL-12,IL-15,NF-?B increased.when the NK cells and DC cells to 1:5 ratio of mixed culture,HEP and HE-HEP can increase the secretion of IL-12 in these five concentrations.The effect of HE-HEP on the secretion of IL-12 from DC cells was higher than that of HEP.The secretion of IL-15 in DC cells was significantly increased at the concentration of 250 ?g/mL,125 ?g/mL and 62.5 ?g/mL.In all concentrations,IL-18 and secretion were increased,while HEP had no significant effect.(3)HEP and HE-HEP could promote the expression of IL-12 mRNA at the concentration of 15.625 ?g/mL to 62.5 ?g/mL when NK cells and DC cells were mixed and cultured with 1:5 ratio.HEP and HE-HEP have the same effect.HEP and HE-HEP could promote the expression of IL-12 mRNA at the concentration of 15.625?g/mL to 250 ?g/mL,and the effect of HE-HEP was better than that of HEP.At 37.25 ?g/mL and 15.625 ?g/mL,the effect of HE-HEP was significantly higher than that of HEP in promoting the expression of IL-15 mRNA.At the concentration of 31.25 ?g/mL and 62.5 ?g/mL,the effect of HE-HEP was significantly higher than that of HEP in promoting the expression of NF-?B mRNA.The expression of TNF-? and NKG2D on NK cells was higher than that of HEP when NK cells and DC cells were mixed with 5:1,and the effect of HE-HEP on NK cells was the same at the same concentration.The expression of HE-HEP was higher than that of HEP at the high concentration,and HEP was superior to HE-HEP at low concentration.The expression of GM-CSF mRNA was significantly increased when the concentration of HE-HEP is 25 ?g/mL and the HEP is 125 ?g/mL.(4)HEP and HE-HEP could enhance the degradation of I?B-? and I?B-? protein,promote the expression of p50 and p65 protein in DCs,and the effect of HE-HEP was stronger than that of HEP.In summary,hydroxy ethylation could significantly improve the interaction and immunological activity of Hericium erinaceus polysaccharides on DC cells and NK cells,and could enhance the activation of NF-?B signaling pathway. |
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