Regulatory Effects Of Plant Polysaccharides On Function Of Murine Dendritice Cells And Macrophage

There are numerous plants in the nature,among which many might be used as immunomodulator agents.It is significant to separate the effective components and study on their biological properties,which will provide scientific functions for their further developments as active ingredients in functional food,or new medicine.Antigen-presenting cells(APCs) whose primary function is to capture,process, and present antigens toward T lymphocytes plays an important role in initiating T cell responses against microbial pathogens and tumors.Dendritic cells(DCs) are the most potent professional APCs with distinct abilities to stimulate na(l|¨)ve T lymphocytes and initiate primary immune responses.Given their central role in controlling immunity, DCs are targets for many situations involving anti-infection or anti-tumor immunologic response.In order to illustrate the possible pharmacological mechanism of those medical functions of Chinese medicines from the aspect of immunology,a screening model of immunological activity of phytochemicals by using DCs as the target cells was established.Effects of CPC,TGP,PL-PS,PLP,isoactoside and acteoside on the phenotypic and functional maturation of DCs were tested by using this screening model.Based on the preliminary results,the effects of PL-PS or PLP on the differentiation,development,maturation,and immigration of DCs were studied.In addition,the activation of PL-PS on macrophage which also belongs to APCs was investigated.The purpose of this study was to make a preliminary investigation of the immunomodulation mechanism of the polysaccharides and to provide theoretical foundation for the exploration of products made from the seeds of Plantago asiatica L..Moreover.the screening model established in our study will provide a new approach for the bioactivity-screening studies.The main research results obtained in this dissertation are concluded as follows:1.Investigated the cytotoxity of PLP.CPC,PL-PS,TGP,acteoside and isoacteoside on DCs precursor.We observed the effects of these extracts on cellular morphology with inverted microscope,used MTT method to determine the inhibitory rate of these extracts on cell growth,and applied flow cytometry to determine apoptosis.We found that a series of concentrations of these extracts had neither reliable effect on cell morphology,nor significant inhibition on cell growth and proliferation.The cytotoxicity gradation of various concentrations of these extracts ranged from 0 to 1 degree.The same results were discovered by apoptosis analyses. Thus,we came to the conclusion that in a certain scope of concentration,the extracts we used have no cytotoxity on DCs precursor,and can be used in the selection-of immunopotentiating agent.2.The screening model of immunological activity by using DCs as target cells was established.Based on the classical literature and the current study,we induced the murine bone marrow to immature dendritic cells by rmGM-CSF and rmIL-4.On day 5,the cells were collected,and CD11c~+ cells were isolated by MACS.The morphology were observed by using inverted microscope,scanning electron microscope and transmission electron microscope.While the expression level of surface molecular on DCs were tested by flow cytometry.The results showed that high purity of DCs can be gained,and the method was stabiliy,which could used for bioactivity-screening studies.3.Investigate the immunological activities of CPC.TGP,PL-PS,PLP, isoactoside and acteoside by using the bioactivity-screening model.We observed the DCs after being stimulated by these extracts with inverted microscopic,and finded that these colonies were covered with many sheet-like processes typical of DCs.The effects of PLP,CPC,PL-PS,TGP,acteoside and isoacteoside on proliferation of dendritic cells in vitro were studied by using MTT assay and the results showed that CPC,TGP.PL-PS and PLP have no effects on the proliferation of DCs in certain concertrations,whereas isoactoside and acteoside could significantly stimulate the proliferation of DCs.Flow cytometer showed that like lentinan,all these extracts origin from plane medicine could enhance the expression levels of MHCⅡon DCs surfaces which gated on CD11c~+ DCs,and decrease the uptake dextran by DCs compared with unstimulated DCs.4.Investigated the effects of PL-PS or PLP on the phenotype and phagocytosis on DCs.Flow cytometroy were used to analyze the expression level of MHC classⅡ. co-stimulate molecule CD80,CD86 and CD40 on DCs.We found that both PL-PS and PLP could enhance the expression level of MHCⅡ,CD80,CD86 and CD40.At the four doses(from 10μg/mL to 200μg/mL) tested,there was a concentration-dependent relationship.The effects of PL-PS on the expresision of CD86 on DCs were higher than the same dose of PLP at the medial and high concentration(p＜0.05).Untreated DCs showed high phagocytosis to FITC-dextran, and after being treated with PL-PS or PLP,the phagocytosis were significantly decreased(p＜0.05),which showed a concentration-dependent relationship at the four doses(from 10μg/mL to 200μg/mL) tested.5.Investigated the effects of PL-PS or PLP on the secretion activity of DCs.The level of NO was detected by NO analyzing Kit.The level of mouse IL-12 p70,IL-10, IL-1βand chemotatic factor RANTES in culture supernatants was determined by sandwich enzyme-linked immunosorbent assay.We found that both PL-PS and PLP could enhance the secretion of Th-1 related cytokine like IL-12 p70 and IL-1β,and this effect was dose-depended.With the IL-10 secretion of DCs,both PL-PS and PLP could inhibit significantly the IL-10 secretion(p＜0.05) and the effect of PL-PS is more significant than the same concentration of PLP(p＜0.01).The secretion of RANTES of DCs was also elevated after being treated with PL-PS or PLP.These results showed that both PL-PS and PLP could induce cellullar immunologic response by affecting the cytokine secretion of DCs.6.Investigated the effects of PL-PS or PLP on the proliferation of T cell stimulated by DCs.Antigen presenting ability to allogeneically naive or syngeneically primed T lymphocytes was examined by the lymphocyte proliferation of mixed lymphocyte reaction(MLR).The result demonstrated that both PL-PS and PLP could increase antigen presenting ability of DCs to allogeneically na(l|¨)ve or syngeneically primed T lymphocytes.Both PL-PS and PLP at high concentration play stimulating effects on the proliferation of T cell when the ratio between T cell and DCs was 10:1.7.Investigated the effects of PL-PS or PLP on the expression level of CCR7 mRNA on DCs.Through seim-quantitation PCR method,the expression of chemotactic receptor CCR7 on DCs surface was analyzed to clarify the molecular mechanism that PL-PS or PLP changes chemotactic activity of DCs.Results show that PL-PS at the concentration of 100μg/mL,PLP at the concentration of 50 and 100μg/mL could increse the expression of CCR7,when compared to the untreated group (p＜0.01).The results showed that both PL-PS and PLP have influence on chemotactic factor receptor of DCs surface.8.Investigate the effect of PL-PS or PLP on cytotoxicity of specific cytotoxic T-lymphocytes(CTL) induced by DCs.Cultured murine DCs were pulsed with SP2/0 tumor cell lysates and co-incubated with PL-PS or PLP.SP2/0 specific CTL were induced by spleen lymphocytes stimulated with DCs.The results showed that both PL-PS and PLP promoted LDH activities released into culture supernatants.Thus,we came to the conclusion that both PL-PS and PLP could promote the cytotoxicity of specific CTL induced by DCs which pulsed with SP2/0 tumor antigen durning the stage of antigen presentation.9.The immunomodulatory activities of PL-PS were examined on RAW 264.7 and murine peritoneal macrophage.By using MTT method,Griess method,flow cytometry and ELISA method,we found that PL-PS could activated RAW264.7 cells to produce cytokines such as tumor necrosis factor(TNF)-α,IL-1β,and nitric oxide (NO)dose dependently.It induced the expression of co-stimulatory molecules such as B7-1,B7-2 and CD40 on RAW264.7 cells.PL-PS could also activated murine peritoneal macrophage s to produce NO and IL-10.These results show that PL-PS possess potent immunomodulatory activity on macrophage lineage cells.The results demonstrated that both PL-PS and PLP could improve the proliferation of T cell and enhance the specific cytotoxity activity of CTL by improving the expression of adhesionand co-stimulating moleculars on the surface of DCs and secretion of Th1 related cytokines.By elevating the exprssion level of CCR7 on DCs,both PL-PS and PLP can enhance the migration of DCs.In addition. PL-PS could enhance the activation of macrophage lineage cells.These results may provide experimental basis of pharmacodynamics for clinical application of PL-PS and PLP.