| ObjectiveDendritic cell (DC) is the most powerful and professional antigen-presenting cell. It is the first line of defence against invading pathogens. With the help of various kinds of pattern recognition receptors (PRR), DC is able to identify microbes, thus making the widely monitoring effect of a certain kind of pathogen. The pattern recognition ability of DC, not only determins the monitoring capacity of the body against pathogen intrusion, but also plays a key role in the intensity and pattern of acquired immunoreaction. Therefore, the diversification of DC phenotype, and the interactions between DC and T cell are the hot topics and key points of infection immunity research. Based on the classical theorys of Ying and Wei, Traditional Chinese medicine has an advantage in Anti-infective and anti-virus treatment, and the reliability of the theory is proved repeatedly in practice. Therefore, the purpose of this study is to investigate pattern recognition of DC, to inspect the interacting characters between DC and T cell, and to observe the regulating mechanisms of a classical Ying and Wei prescription Yu-ping-feng-san(YPF) on pattern recognition ability of DC and T cell differentiation.Methods:1. Preparation of YPF: YPF decoction was prepared by water extracting and alcohol precipiting according to the classical methods.2. DC cultures: Bone marrow mononuclear cells were isolated from Balb/c mice under sterile condition with traditional methods, induced to form DCs by GM-CSF and IL-4 in vitro. Then detect the immature DC phenotype feature and the expression of pattern recognition receptors(TLR3,TLR4,TLR5,TLR7,TLR9).3. Flow cytometry was used to observe of DC phenotype after stimulation with TLRs ligands (pathogen analog molecules), such as CD11c, antigen-MHC class II complexes and costimulatory molecules CD80, CD86.4. RT-PCR technique was applied to examine the transcriptional levels of TLR3, TLR4, TLR5, TLR7, TLR9, CD86, CD80, CD40, CCR7 and IL-12p40.5. To observe DC stimulate T cell differentiation with and without TLRs ligands activation and YPF treatment using DC-T cell co-culture method.Results:1. TLRs ligands can stimulate DC maturation and induce high-expression of costimulatory moleculesStimulation of DC with TLRs ligands induced an increase in the percentage of cells expressing of MHC-Ⅱ,CD80 and CD86(P<0.05). LPS induced a most significant increase(P<0.05). Compared with LPS stimulation group, different TLRs synergy group DC shows higher expression of MHC-Ⅱ, CD80 and CD86. Poly(I:C) and LPS stimula- tion group were highest.2. LPS-PolyI:C synergy stimulate higher expression of DC function factorRT-PCR result showed: compared with control group, Poly(I:C) and LPS single stimulation group, CCR7 and IL-12P40 mRNA were up-regulated; In Poly(I:C) and LPS synergistic stimulation group, the expression of CCR7 and IL-12P40 mRNA were significant increased.3. YPF promote DC proliferationIn DC culturing system,YPF showed significant promoting effect of DC proliferation on iDC(CD11c+I-Ad-), for the proportion as well as the absolute quantity of CD11c+I-Ad- elevated obviously(P<0.05), MHC-Ⅱ,CD80,CD86 decreased comparatively. In LPS-PolyI:C synergy stimulating system, YPF can further promote the maturity of DC, increasing the expressing levels of signaling molecules such as MHC-Ⅱ,CD80,CD86 etc.4. LPS stimulated DC influence the differentiation of T cell subpopulationLPS stimulated DC induced secretion of Th1 cytokines IFN-γ, inhibit IL-10 secretion. LPS and polyI:C synergy stimulated DC promote Th1 type cytokines to secrete IFN-γand IL-17; LPS and Flagellin synergy stimulated DC can decrease IFN-γ, IL-17 secretion, but significantly increase IL-10 secretion.5. Influence of YPF & LPS stimulated DC synergy on the differentiation of T cell subpopulationYPF and LPS synergy stimulated DC increased the secretion of IFN-γ, IL-17 and IL-10, had no effect on the secretion of TNFα. YPF, LPS and polyI:C synergy stimulated DC decreased the secretion of IFN-γand IL-17, increased the secretion of TNFαand IL-10. YPF, LPS and Flagellin synergy stimulated DC increased IFN-γsecretion significantly while inhibiting TNF-α,IL-17 and IL-10 secretion.Conclusions:1. TLRs ligands can stimulate DC maturation and induce high-expression of costimulatory molecules and migration-factor.2. Compared with LPS group, TLRs ligands synergy group can stimulate higher expression of DC maturity and function factor.3. YPF can promote immature DC proliferation, in synergy with TLRs ligands enhanced mature DC; can attain higher expression of antigen presentation molecular; can costimulate molecules; can adjust information transmission between DC and T cells, and also can induce antivirus factor IFN-γand IL-10 expression, reduce inflammatory factor TNFαand IL-17 secretion.Therefore, YPF enhance DC mature and raise the expression of costimulatory signal, meanwhile, adjust information transmiting ability as well as and transmiting microenvironment between DC and T cells, by promoting antiviral ability and anti-inflammatory effect of T cells, eliminating pathogens, controling the development of inflammation, thus improved the pathogens resisting ability of the body. |
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