Study On A Certain Purity Of Traditional Chinese Medicine Compound Polysaccharides With Different MHC B-LβⅡ Genotype Of Chicken Immunomodulatory Effects Receptor TLR2,TLR4 And Immune Signaling Molecule

In this study, we used the certain purity of traditional Chinese medicine compound polysaccharide（cCHMPS） which has proven to to be a significant immune-enhancing effect,（content 77.1%）,acting on different MHC B-LβⅡ genotype chicken peripheral blood lymphocytes in vitro to determine the（cCHMPS）immune regulation dose would be affected MHC B-LβⅡ gene polymorphism,, and clear the Immunomodulatory effects receptor of cCHMPS.Study the immunomodulatory role of cell signaling molecules. Provide a theoretical basis for cCHMPS which would develope into Immunoenhancer.Method:1.PCR-SSCP technique was applied to analyze the polymorphism of MHC B-LβⅡ gene in Huang-Ma chickens.,separated the chicken with different genotypep.Collection peripheral blood,isolation and culture of lymphocytes with different puritied cCHMPS.Then detecting the expression of lymphocyte MHC B-LβⅡ mRNA using Real-time PCR method after 24 h.2.Collected the different MHC B-LβⅡ genotype from Huang-Ma chickens,isolated peripheral blood lymphocytes, with a final concentration of 0.5 μg / mL, 1 μg / mL, 2 μg / mL of TLR2, TLR4 antibody after co-culture 1h, then added to cCHMPS which the final concentration is 200 μg/mL,100 μg/mL,50μg/mL,25 μg/mL, 0 μg/mL co-cultured 24 h, using the ELISA method to detect the content of IL- 6, TNF-αand NF-κB in lymphocyte culture supernatant.3.Collection the lymphocytes from Huang-Ma chickens, isolated peripheral blood,and added to cCHMPS which the final concentration is 200 μg/mL,100 μg/mL, 50 μg/mL,25 μg/mL,0 μg/mL co-cultured 1h,2h, 4h,detected the Lymphocyte supernatant cAMP and cGMP content by ELISA. And Ca2 +,NO, iNOS content by ELISA when 24 h.Result:1.After digestion the MHC B-L βII gene exon 2,we found four alleles and five genotypes. In cCHMPS group and the control group, the MHC B-LβⅡ mRNA expression levels in AA, AB genotype were higher than BB, AC, AD genotype.2.The genotype chicken lymphocytes only polysaccharide processing, secretion of cytokines IL-6,TNF-α,NF-κB was increased compared to the level of the control group（P<0.05）; co-culture with TLR2,TLR4 antibody and then incubated with cCHMPS,the promotion of cCHMPS lymphocytes secrete IL-6, TNF-α and NF-κB synthesis was inhibited, and with the increase of the concentration of antibody,inhibition increased.3.Polysaccharide group compared with the control group, were significantly increased in the supernatant of cAMP, cGMP levels（P <0.05）. But there are differences in their secretion with the different times of culture. Compared with the control group, the group of cCHMPS can promote the secretion of NO,iNOS,Ca²⁺.Different polysaccharide concentration of each cytokine secretion amount corresponding to the highest.Conclusion:1.Individual genetic differences MHC B-LβⅡ polymorphism caused to some extent, affected the cCHMPS immune-enhancing optimum dose.2.cCHMPS was combined with the cell surface TLR2/TLR4 Acceptor,by TLRs/NF-κB signaling pathway activation of the transcription factor NF-κB,regulatory cytokine IL-6,TNF-α synthesis,promoting the secretion of Cytokines,such as,IL-6,TNF-α,Ca²⁺,NO,iNOS.Start the immune cell signaling pathways and exert the Immunomodulatory effect.