| Traditional Chinese medicine stress personalized medicine since ancient times, the development ofgenomics provides theoretical support for personalized medicine. The genomics research applicated in thefield of drug treatment began to pop up when the interaction between genetic variation and drug efficacywas realized. Polysaccharides extracted from compound Chinese herbal medicinal can enhance immunity,and some research has shown that polysaccharides can be not only presented to T cells receptor by MHCclassⅡmolecules, activated the adaptive immune response, but also effectively activate B lymphocytes andinduce production of specific antibodies. The polymorphism of chicken major histocompatibility complexgene (MHC) enables different individuals having different immune responses to the same antigen, andleading to differences in immune function, which may affect the screening of compound Chinese herbalmedicinal polysaccharides (cCHMPS) optimum immune dose. Meanwhile, the immunoregulationmechanism of polysaccharides extracted from compound Chinese herbal medicinal is not clear.Object:1.Study the effects of the puritied cCHMPS, which has been proven to significantly effect on theimmunity of mice, on immune function and select its best dose.2.Study the effects of MHC B-LβII gene exon2RFLP polymorphism on immune function and theeffect of puritied cCHMPS on immunity in different MHC B-LβII genotype chickens, explore whether thepolymorphism of MHC B-LβII gene affect the screening of cCHMPS immune-enhancing doses.3.To study whether TLR2is the pattern-recognition receptor of cCHMPS, which act on the cellsurface TLR2receptor, and then induced lymphocyte proliferation and IL-12secretion.4.To investigate the effects of cCHMPS on the contents of the second signal molecules cAMP'cGMP in lymphocytes.Methods:1.The chicken peripheral blood among physiologic saline, levamisole hydrochloride, astragaluspolysaccharides(APS) and high, medium and low-dose puritied cCHMPS group was collected on days8,14,21,28,35and42after the first vaccination.Then they were sampled for determination of T, Blymphocyte proliferation, ND antibody level, IFN-γ、IL-4、IL-12contents, SOD activities and immunityfunction of red blood cell by MTT and ELISA method.2.PCR-RFLP technique was applied to analyze the polymorphism of MHC B-LβII gene exon2inmale liangfeng Green-Feet Ma chickens.3.The blood was collected on days8,14,21,28,35and42after the first vaccination.The effects ofMHC B-LβII gene exon2and puritied cCHMPS at different dose on T、B lymphocytes proliferation, NDantibody level, IFN-γ、IL-4、IL-12contents and their mRNA expression were evaluated by MTT, ELISAand fluorescence real time quantitation PCR (RT-PCR).4.Chicken peripheral blood lymphocytes with cCHMPS at the final concentration of50μg/mL werecultured with different concentration of TLR2antibody, specific NF-κB blocker PDTC in vitro for48h, respectively, then the cell supernatants in different antibody and blocker were analyzed for changes incontents of IL-12.5.Chicken peripheral blood lymphocytes were cultured with cCHMPS at the final concentration of50μg/mL in vitro for20min,1,2,4,8h, respectively, then the cells were collected to determine the contents ofcAMP and cGMP by ELISA.Results:1.The T, B lymphocyte proliferation, IFN-γ, IL-4and IL-12contents, SOD activities and immunityfunction of red blood cell astragalus in APS and cCHMPS at different dose groups were higher than that ofcontrol group, in levamisole hydrochloride group was higher than that of in control group, but there was nosignificant difference (p>0.05). Among them, the immune enhancement effects of medium-dose cCHMPSwas the best.2.A, B two alleles and AA，AB and BB three genotypes were detected by digestion of restrictionendonuclease Hin1I in MHC B-LβII gene exon2, the lymphocyte proliferation, ND antibody level, IFN-γand IL-4contents of genotype AA，AB were higher than that of genotype BB; there were no significantdifferences (p>0.05) between AA，AB and BB type in the concentration of IL-12.3.The puritied cCHMPS concentration and MHC B-LβII gene polymorphism have significant effectson the immune traits, but the interaction between the puritied cCHMPS concentration and MHC B-LβIIgenotype has significant influence on T, B lymphocyte proliferation, the ND antibody level and IFN-γcontents; there were different of the most optimum cCHMPS dose to increase immunity in different MHCB-LβII genotype chidkens, the B lymphocyte proliferation and ND antibody concentration of AA genotypewas higher than that of BB genotype in high-dose puritied cCHMPS, the T, B lymphocyte proliferation andND antibody concentration of AB genotype was higher than that of AA genotype in middle-dose puritiedcCHMPS, the T lymphocyte proliferation and ND antibody level of AB genotype was higher than that ofBB genotype in low-dose puritied cCHMPS, the dominant genotype of IFN-γ in each dose cCHMPS groupwere AB genotype.4.The stimulatory effect of cCHMPS on the production of IL-12was inhibited by anti-TLR2antibodyand PDTC, with the increase of anti-TLR2antibody and PDTC concentration, the inhibitory effectincreased significantly.5.cCHMPS could improve the cAMP and cGMP contents of chicken peripheral blood lymphocytecultured in vitro.Conclusion:1.The immune enhancement effects of medium-dose cCHMPS was higher than levamisolehydrochloride, APS and high, low-dose cCHMPS group.2.The genetic differences between individuals caused by MHC B-LβII gene Hin1I locuspolymorphism may partly affectded the screening of cCHMPS optimum immune dose, the optimumimmune dose of cCHMPS slightly different in different genotype groups. 3.The mechanism of cCHMPS promoting lymphocyte proliferation and IL-12secretion might be asfollows: the cCHMPS act on the cell surface TLR2receptor, and then activate TLR2-NF-κB signaltransduction pathway, change the signaling molecules cAMP and cGMP contents, make the protein in thesignal path phosphorylated and have biological activity, activate the nuclear transcription factor NF-κB atlast, which contribute to regulate the expression and transcription of IL-12. |
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