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表达弹性蛋白酶抑制因子的益生菌大肠杆菌Nissle 1917可预防实验性结肠炎

[Elafin-expressing probiotic Escherichia coli Nissle 1917 protects against experimental colitis].

作者信息

Liu Z L, Wang W H, Liu Y, Wu T, Teng G G

机构信息

Department of Gastroenterology, Peking University First Hospital, Beijing 100034, China.

出版信息

Zhonghua Yi Xue Za Zhi. 2021 Dec 14;101(46):3819-3824. doi: 10.3760/cma.j.cn112137-20210318-00689.

Abstract

To construct the gene modified probiotic Escherichia coli nissle1917 (EcN) which can express human Elafin protein and to explore its protective effect on the acute colitis in mice. The recombinant plasmid with human gene was constructed and then transferred to EcN. Western blot results confirmed that the engineered probiotic expressed Elafin successfully in vitro. C57/BL6J mouse was used in this study and were randomly divided into 4 groups according to different treatment: PBS gavage (PBS group); DSS administrated (DSS group); DSS administrated with wild-type EcN (EcN-WT) gavage (EcN-WT group); DSS administrated with EcN-Elafin gavage (EcN-Elafin group). Body weight and disease activity index (DAI) were measured every day. The length of mice colons in each group were measured after euthanasia. The degree of inflammation of intestinal mucosa in each group was measured through histopathological scoring. The proportion of neutrophils and macrophages infiltrated into colon lamina propria was detected by flow cytometry. The protein expression levels of pro-inflammatory cytokines TNF-α, IL-6 and chemokine CXCL-1 in colonic tissue were quantified by enzyme-linked immunosorbent assay. Elafin protein could be detected in the supernatant of EcN-Elafin culture medium and EcN-Elafin homogenates. Compared with DSS group, the weight loss and DAI score of EcN-Elafin group and EcN-WT group were both significantly improved. The colon length of EcN-Elafin group was significantly longer than that of DSS group. The histological score of colitis in EcN-Elafin group was significantly lower than that in DSS group (5.3±2.3 vs 9.3±1.4, <0.05). In EcN-Elafin group, the proportion of neutrophils[(8.65±1.49)% vs (17.60±2.16)%, <0.01]and macrophages[(3.79±0.26)% vs (5.73±0.45)%, <0.01]infiltrated into the colon lamina propria was significantly decreased compared with DSS group. The protein expression levels of TNF-α, IL-6 and CXCL-1 in EcN-Elafin group and EcN-WT group were significantly lower than those in DSS group. Elafin-expressing EcN can protect against DSS-induced acute colitis in mice and may have provided an effective and cost-efficient method for the treatment of IBD.

摘要

构建可表达人弹性蛋白(Elafin)的基因工程益生菌大肠杆菌Nissle1917(EcN),并探讨其对小鼠急性结肠炎的保护作用。构建含人Elafin基因的重组质粒,然后将其转入EcN。蛋白质免疫印迹结果证实该工程益生菌在体外成功表达了Elafin。本研究使用C57/BL6J小鼠,根据不同处理将其随机分为4组:灌胃PBS(PBS组);给予葡聚糖硫酸钠(DSS)(DSS组);给予DSS并灌胃野生型EcN(EcN-WT组);给予DSS并灌胃EcN-Elafin(EcN-Elafin组)。每天测量体重和疾病活动指数(DAI)。安乐死后测量每组小鼠的结肠长度。通过组织病理学评分测量每组肠黏膜的炎症程度。采用流式细胞术检测浸润到结肠固有层的中性粒细胞和巨噬细胞比例。采用酶联免疫吸附测定法定量结肠组织中促炎细胞因子TNF-α、IL-6和趋化因子CXCL-1的蛋白表达水平。在EcN-Elafin培养基上清液和EcN-Elafin匀浆中可检测到Elafin蛋白。与DSS组相比,EcN-Elafin组和EcN-WT组的体重减轻和DAI评分均显著改善。EcN-Elafin组的结肠长度显著长于DSS组。EcN-Elafin组结肠炎的组织学评分显著低于DSS组(5.3±2.3对9.3±1.4,<0.05)。与DSS组相比,EcN-Elafin组浸润到结肠固有层的中性粒细胞比例[(8.65±1.49)%对(17.60±2.16)%,<0.01]和巨噬细胞比例[(3.79±0.26)%对(5.73±0.45)%,<0.01]显著降低。EcN-Elafin组和EcN-WT组中TNF-α、IL-6和CXCL-1的蛋白表达水平显著低于DSS组。表达Elafin的EcN可保护小鼠免受DSS诱导的急性结肠炎,可能为炎症性肠病的治疗提供了一种有效且经济的方法。

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