Research For The Inflammation Improvement Effect And Immunoregulation Mechanism Of Recombinant Lactobacillus Expressing IL-10 Combined With 5-aminosalicylic Acid On Dextran Sulphate Sodium-induced Colitis Mice Background And Purpose

Research for the inflammation improvement effect and immunoregulation mechanism of recombinant Lactobaciilus expressing IL-10 combined with 5-aminosaIicylic acid on dextran sulphate sodium-induced colitis mice Background and purposeInflammatory bowel disease(IBD), including ulcerative colitis and crohn'disease, is an agnogenic,recurrent and chronic intestinal disease. The incidence of IBD has been greatly increased in the past 20 years.Lactobacillus plays an important role in promoting the intestinal flora balance, regulating immune function and enhancing intestinal mucosal barrier and pathogen defense. Interleukin-10 (IL-10), also known as cytokine synthesis inhibitory factor(CSIF), is a typical anti-inflammatory and immunosuppressive cytokines, which plays an important role in the development of inflammatory bowel disease and in long-term survival of Treg such as CD4+ and CD25+, in maintaining inhibition mechanism and induction of inducible Treg cells.We integrated IL-10 gene into the genome of L.casei and detected the objective protein, then the recombinant lactobacillus had been used on the treatment of dextran sodium sulphate -colitis mice, at last we observed the inflammation improvement effect of and discussed the mechanism. Methods:Part 1 The construction of a recombinant lactobacillus strain expressing interleukin-10A signal peptide sequence was synthesized and cloned into the integrative plasmid pIlac down-stream of lactose-inducible promoter. The recombined plasmid pIlac-sp-IL10 was then introduced into Lactobacillus casei CECT 5276 by electroporation, PCR using the genomic DNA of the recombinant lactobacillus as template was performed to confirm the the integration of IL-10 gene into the chromosome. Western blot was used to detect the expressing of the recombinant protein.Part 2 Research for the inflammation improvement effect and primary mechanism of recombinant lactobacillus expressing IL-10 combined with 5-aminosalicylic acid on dextran sulphate sodium-induced colitis mice.The mice is divided into 7 groups, including normal control group, DSS model group, DSS model+5ASA treatment group, DSS model+ lactobacillus group(broth with 2×108/ml)+ 5ASA treatment group, DSS model+ low dose transgenic lactobacillus by IL-10(broth with 2×107/ml)+5-ASA treatment group, DSS model+ medium dose transgenic lactobacillus by IL-10(broth with 2×108/mL) +5-ASA treatment group, DSS model +high dose transgenic lactobacillus by IL-10 (broth with 2×109/mL)+5-ASA treatment group.All the experimental mice were killed at the 11th day, and we observed the changes including colon length, DAI, HS and HE, etc. Real-time PCR were used to detect the levels of NF-κB, PPAR-γ, IFN-γ, TGF-β, IL-10 in peripheral blood and ELISA were used to detect the levels of PPAR-β, IFN-γ, TGF-β, IL-10 in colon tissue of mice Results:Part 1The IL-10 gene had been integrated into the genome of L. casei, and the objective protein was partially secreted into the supernatant.Part 21 With the same treatment concentration (2*108CFU/ml), the recombinant lactobacillus had a better curative effect than the common lactobacillus.2 Among the three different concentration (2*107CFU/ml,2*108CFU/ml, 2*109CFU/ml) of the recombinant lactobacillus,2*109CFU/ml had the best treatment effect.Conclusion:The recombinant lactobacillus had a certain treatment effect on DSS-colitis mice.