Effect Of Polysaccharide Isolated From Fruit Of Physalis Alkekengi L. On The Phenotypic Maturation Of Murine Bone Marrow Derived Dendritic Cells And Analysis Of Its Immune Receptor

In recent years, with the development of the research on plant polysaccharides,it was found that a lot of plant polysaccharides have the function of imunoregulation.Our group has studied on the adjuvant effect of PPSB,the results show that,PPSB have significant immunologic enhancement in candida albicans nucleic acid vaccine,however, little is known about the mechanisms of immunologic enhancement induced by PPSB.Studies have shown that, adjuvant assisted antigen stimulating the body’s immune response almost all related to the role of dendritic cells (DC). The maturing of the DC is a key step to initiate specific immune responses,while expression of Toll-like receptors(TLRs) on DC is a bridge connecting natural immune and adaptive immune responses.With DC as the breakthrough point, TLR4as target,this dissertation discussed the mechanism of immunologic enhancement induced by PPSB preliminarily.The main research results obtained in this dissertation are concluded as follows:1.Established the generation of murine bone marrow dendritic cells(BMDC)technology system in vitro.Two methods were used to generate BMDC,and the morphology were observed by using inverted microscope, the expression level of surface moleculars on BMDC were tested by flow cytometry.The results show that, we induced the murine bone marrow cells to BMDC by rmGM-CSF alone,on day8,only collected a low ratio of BMDC,while we induced the murine bone marrow cells to BMDC by rmGM-CSFand rmIL-4,on day8, a large number of high ratio of immature BMDC could be gained,therefore we use rmGM-CSFand rmIL-4inducing method, and collected the immature state of BMDC on day5for subsequent experiments.2.PPSB enhanced the expression level of co-stimulatory molecules CD80andCD86on BMDC.To further investigate the effects of PPSB on the phenotype of BMDC,we treated BMDC with different concentrations of PPSB,then flow cytometroy were used to analyze the expression level of co-stimulatory molecules CD80and CD86on BMDC. The results show that,compared with the control group,we found that both PPSB could enhaned the expression level of CD80and CD86to varying degrees, in the range of concentration from lOug/ml to50ug/ml, there was a concentration-dependent relationship, therefore.PPSB could modulate the phenotypic maturation of BMDC.2.PPSB could inhibit the binding of LPS to the cell surface of BMDCTo further investigate the specific binding of PPSB on BMDC.different concentrations of PPSB were used to inhibit the binding of FITC-LPS to TLR4on BMDC,unlabeled LPS as a background control,and flow cytometroy analysis the binding of FITC-LPS to BMDC.We found that different concentrations of PPSB could inhibit the binding of FITC-LPS,and with the increase of the concentration of PPSB, the effect of inhibition was obvious gradually,implying PPSB binding to the surface of BMDC via TLR4.