| Lactic acid bacteria exopolysaccharides (LAB EPS) had unique physical a chemical nature, meanwhile had a variety of biological activity. L. casei was widely used in yogurt, cheese and other foods widely, making the EPS to be able to play much physiological activity, at the same time there are many not yet clear, causing the research interests of scientists. Dendritic cells (dentritic cell, DC) is currently known as the strongest function of antigen-presenting cells in vivo. DC is a major immune response initiator playing a key role in the induction of immune responses. In this study we investigated the effect of EPS on the phenotypic and functional maturation of in intestinal lamina propria and BMDC, to clarify the immunomodulatory mechanisms of Lactobacillus casei exopolysaccharides.This study was mainly conducted in the following aspects.(1) The EPS was extracted from the fermentation broth of Lactobacillus casei.(2) Murine bonemarrow derived dendritic cells (DMDCs) were acquired by cytokin-induced method.(3) The cell morphological were examined under optical microscope, flow cytometry (FACS) analyzed the surface molecules of DCs in intestinal lamina propria CDllc, IA/IE and costimulatory factor CD80expression.(4) The secreted chemokine and chemokine receptor of DCs treated with different doses of EPS were detected. In vivo, the level of mouse IL-12p70, FKN and MIP-3a in serum was determined by sandwich enzyme-linked immunosorbent assay. In vitro, The level of mouse IL-12p70, CCR6and CX3CR1in culture supernatant was determined by sandwich enzyme-linked immunosorbent assay.(5) Impact of EPS on transcription level of DCs IL-12mRNA, chemokine mRNA and its receptor mRNA were detected. Transcription level of chemokine mRNA of DCs in intestinal mucosa lamina propria transcription level of mouse bone marrow-derived DCs chemokine receptor mRNA and IL-12mRNA were determined by RT-PCR.(6) Impact Of EPS on DCs subtypes of intestinal lamina propria. Changes in the number of CD103+CD11b+DCs in the lamina propria of the intestinal mucosa and CD103-CDllb+DCs were analyzed by flow cytometry.(7) Impact Of EPS on histone H3acetylation levels of IL-12gene promoter in bone marrow-derived DCs were detected. Histone H3acetylation levels of IL-12p35and IL-12p40gene promoter was detected by chromatin immunoprecipitation kit.Results were showed as follows:(1) EPS could increase the level of IL-12p70, FKN and MIP-3α in serum, the effect reached the maximum when EPS concentration was50mg/kg.(2) EPS could contribute to bone marrow derived DCs secrete IL-12p70, CCR6and CX3CR1, and the effect expressed dose-dependent.(3) DCs by flow cytometry the surface molecule CD11c, IA/IE and costimulatory factor CD80expression. Comparing to the mouse that only subjected to Saline, the expression of CDllc, IA/IE and co-stimulatory factor CD80by DCs in intestinal mucosa lamina propria with EPS stimulation were increased.(4) EPS could increase the transcription level of FKN mRNA and MIP-3a mRNA in DCs in the intestinal lamina propria.(5) EPS could increase the transcription level of CCR6mRNA, CX3CR1mRNA, IL-12p35mRNA and IL-12p40mRNA in bone marrow-derived DCs.(6) EPS could increase the number of CD103+CD11b+DCs and reduce the number of CD103"CDllb DCs in the lamina propria.(7) EPS could increase the acetylation level of IL-12p35and IL-12p40gene promoter in bone marrow-derived DCs. Thus, EPS could promote the maturation and migration of DCs.Conclusion were showed as follows:EPS could contribute that DCs in blood migrated to the intestinal lamina propria, and the effect was regulated by chemokine; EPS could promote the maturation of DCs in the intestinal lamina propria and could increase the number of CD103+CD11b+DCs in the intestinal lamina propria, EPS could increase the expression level of IL-12gene in bone marrow-derived DCs by epigenetic pathway. |
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