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Treatment of Murine Colitis by IL-10 Gene-transformed Escherichia Coli

Author: GuQiuPing
Tutor: BaiAiPing
School: Nanchang University
Course: Internal Medicine
Keywords: mIL-10 Transformation E. coli Biological activity IL-10 Colitis Inflammatory bowel disease Gene therapy
CLC: R574.62
Type: Master's thesis
Year: 2011
Downloads: 18
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Abstract

The first part of the IL-10 gene of E. coli Objective: murine interleukin-10 gene into E. coli, the bacteria transformed with the expression and secretion of IL-10. Methods: According to mIL-10 gene sequences and E. coli vector cloning site, optimized modified mIL-10 gene sequence, synthesize new mIL-10 gene sequence, connected to the prokaryotic expression vector pET32a and sequenced, EcoR Ⅰ and Hind Ⅲ, double restriction endonuclease gene fragment size. Using genetic engineering techniques containing mIL-10 gene sequence pET32a / mIL-10 was cloned into the prokaryotic expression vector Origami B (DE3), in vitro induced by IPTG, choose the best inducing conditions. Western blot for protein identification and explore IL-10 on LPS-induced activation of RAW264.7 cells. Results: Optimized mIL-10 gene sequence homology at the amino acid translation is 100% recombinant prokaryotic expression vector OrigamiB (DE3) / pET32a mIL-10 can be efficiently expressed in vitro, the optimal induction time IPTG 1mmol induced 3 hours maximum expression, bacterial culture medium the expression of the target protein can be detected, the concentration of 3.96ng/ml. Western blot detection of protein antigens have good reactivity, IL-10 can inhibit the activation of LPS on RAW264.7, reducing the release of TNF. Conclusion: mIL-10 expression in E. coli can have biological effects IL-10. The second part of the IL-10 gene into E. coli colitis in mice Objective: To study IL-10 gene by E. coli transformed with DSS colitis in mice against intestinal inflammation, and to explore the underlying mechanism. Methods: IL-10 gene into E. coli referred to as E.coli-IL-10, empty plasmid transformed bacteria are referred to as E.coli0; The mice were randomly divided into six groups: normal control groups, DSS group, DSS E .coli/IL-10 group, DSS E.coli0 group of normal mice and normal mice E.coli E.coli/IL-10 group group. Establish a mouse model of acute DSS colitis. Since the establishment of the first mouse model of day 1, DSS E.coli/IL-10 E.coli/IL-10 group and normal mice were treated with recombinant IL-10 E. 1 × 108cfu / day orally to the end of the experiment, DSS E . coli and normal mice were treated with empty plasmid E. coli E.coli gavage to end of the experiment, the normal control group and DSS group was given orally to the culture medium at the same end of the experiment. Each group were observed daily disease activity index (DAI), and after the end of the experiment were assayed inflammatory bowel tumor necrosis factor (TNF) and myeloperoxidase (MPO) and other content, and measuring nuclear factor mouse colon (NF)-KB P65 expression. Results: 1.DAI Rating: DSS-E.coli/IL-10 DAI score of mice with DSS and DSS-E.coli0 group scored significantly lower compared to the two groups (P lt; 0.05), DSS-Ecoli0 group DSS group with no significant difference between the DAI score (P gt; 0.05); control group with normal mice Ecoli0, normal mice Ecoli/IL-10 DAI score between the three groups were 0.2. histological score: DSS-Ecoli / IL-10 (6.22 ± 3.30) than DSS group and the DSS-Ecoli0 [(10.54 ± 4.15) and (10.0 ± 3.00)] (P lt; 0.05), after the score was no significant difference between the direct (P gt; 0.05 ); DSS drinking water in each group were higher (0.88 ± 0.31) (P lt; 0.05). 3.MPO activity: DSS E.coli/IL-10 group (2.35 ± 0.39) was significantly lower than the DSS group and DSS Ecoli0 group [(4.15 ± 0.77) and (3.5 ± 1.23)] (P lt; 0.05), higher than the other three groups (P gt; 0.05). 4 tissue TNF in: DSS group (237.85 ± 47.01) and DSS-Ecoli0 (239.81 ± 50.38) was no significant difference between the groups (P gt; 0.05), higher than the other four groups (P lt; 0.05); DSS -Ecoli/IL-10 group (172.46 ± 66.71) than DSS group and DSS-Ecoli0 (?) and (P lt; 0.05), higher (P gt; 0.05). 5 colonic NF-kB expression: DSS-Ecoli/IL-10 mice mucosa of NF-kB activity was significantly lower than the DSS group and the DSS-Ecoli0 group (P lt; 0.05), DSS drinking water in three small groups Rat mucosal NF-kB activity was significantly higher than the three groups of mice drinking tap water with high activity, DSS group and DSS-Ecoli0 NF-kB activity between the two groups had no significant difference (P gt; 0.05). Conclusion: The IL-10 gene into E. coli can significantly alleviate the damage DSS colitis in mice, reduced MPO activity, inhibition of inflammatory bowel inflammatory cells of NF-kB activation and inflammatory cytokine secretion. Use of genetic engineering technology combined with intestinal commensal bacteria expressing IL-10 treatment of IBD can provide a new approach.

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