Treatment Of Murine Colitis By IL-10 Gene-transformed Escherichia Coli

Part I The research on transformed Escherichia coli with IL-10 geneObjective:To successfully transform E.coli with murine IL-10 geneMethord:A murine IL-10 (mIL-10) gene fragment is inserted into the prokaryotic expression vector pET32a after the gene sequence and the plasmid were digested with endonucleases spot Ecoi I and HindⅢ, respectively. The sequence of recombinant plasmid pET32a/IL-10 was confirmed by endonucleases digestion, and then transformed into E. coli Origami B (DE3).The recombinant plasmid transformed host cell strain E. Coli Origami B (DE3) is induced by low IPTG at 37℃to produce the interest protein. The expressed fusion protein was identified with SDS-PAGE and Western blotting. Amount of IL-10 was assayed with ELISA and its bioactivity was analyzed by TNF production inhibition of LPS-stimulated RAW264.7 cells induced by IL-10 secreting E. coli culture medium.Result:The constructed recombinant plasmid pET32a/IL-10 was identical with the reported one,which was identified by endonucleases digestion and sequence.The molecular weight of soluble fusion IL-10-Trx is 34,300,which is detected by SDS-PAGE and WB.The IL-10-Trx recombinant protein was detected in the supernatant of bacteria culture medium, by which significantly inhibited TNF production of LPS-treated RAW264.7.ConcIusion:The The recombinant mIL-10 protein with high bioactivity is expressed by IL-10 gene-transformed Escherichia coli, PartⅡTreatment of murine colitis by IL-10 secreting Escherichia coliObjective:To study the therapeutic effect of IL-10 secreting escherichia coli on murine colitis.Methods:The IL-10 gene-transformed Escherichia coli and empty plasmid transformed Escherichia coli were named E.coli-IL-10 and E.coliO respectively. Mice were divided into six groups:control,DSS, DSS+E.coliO,DSS+Ecoli/IL-10, E.coliO and E.coli/IL10. The mice in groups DSS, DSS+E.coliO,DSS+Ecoli/IL-10 were fed with 5%DSS(w/v) solution for 7 days to induce colitis, the other groups were received normal tap water.As the same time,different medications were given orally as following:(1)DSS+EcoliO and EcoliO:administration of E.coliO;(2) DSS+Ecoli/IL-10 and Ecoli/IL-10:administration of recombinant E.coli/IL-10; (3)DSS and control:administration of LB(Luria-Bertain), each medication was administration from the frist days of the experiment to the end of experiment.DAI was recorded everyday.The expression of TNF,NF-κB p65 and myeloperoxidase (MPO) in inflamed colon of each group was measured at the end of experiment.Results:1. The DAI of DSS+E.coli/IL-10 is lower than that group DSS and DSS+E.coliO (P<0.05), but no difference within latter two groups (P>0.05);the DAI of group control,E.coliO and E.coli/IL-10 is 0.2. The histologic score of group DSS-Ecoli/IL-10 (6.22±3.30) is lower than group DSS (10.54±4.15) and DSS-EcoliO (10.0±3.00) (P<0.05), while higer than contrl(0.88±0.31),E.coliO(1.22±0.43)and E.coli/IL-10(1.0±0.40)(P<0.05)。There no difference within the groups of DSS and DSS+Ec.oliO (P>0.05).The score of that gorups fed with DSS were higher than that groups received tap water(P<0.05). 3. The MPO activity in colonic tissue of group DSS-Ecoli/IL-10 (2.35±0.39) is lower than groupDSS(4.15±0.77)and DSS-EcoliO(3.5±1.23)(P<0.05), higher than the another 3groups (P>0.05); but no difference within the group of DSS and DSS+E.coliO (P>0.05);4. The levels of TNF in colonic tissue of group DSS (237.85±47.01) were increased significantly while compared with the groups of normal control (18.08±19.6),E-coliO (46.96±22.88) and E.coli/IL-10 (24.38±16.08) P<0.05).5. The NF-kB activity of in mice administration of DSS+E.coli/IL-10 were significantly lower than in those mice in group of DSS and DSS+E.coliO (P<0.05), but no difference between the latter two groups.Conclusion:Local delivery of IL-10 gene-transformed escherichia coli ameliorates DSS-inducd murine colitis, with decreased proinflammatory cytokine productions, inhibited NF-kB activation. Gene therapy strategies using engineered E.coli encoding immunoregulatory cytokines may provide a potential approach for IBD treatment.