Toll样受体5与黏附侵袭性大肠杆菌LF82在炎症性肠病发病中的作用研究

发布时间：2018-04-13 00:36

本文选题：炎症性肠病 + 肠道细菌　；参考：《第四军医大学》2014年博士论文

【摘要】：【背景和目的】近年来炎症性肠病（inflammatory bowel disease，IBD）的发病一直呈上升趋势，但是病因不明，发病机制尚不清楚，且难以彻底治愈，严重影响生命健康。一般认为感染、环境等因素触发了遗传易感个体异常的肠道免疫反应，引起肠道持续的炎症和组织破坏。肠道菌群作为重要的致病因素，直接或间接地参与了包括IBD在内的诸多消化系统疾病的发生发展。其中黏附侵袭性大肠杆菌（adherent-invasiveEscherichia coli，AIEC）是近年来受到广泛关注的一种IBD可能致病菌，最具代表性的AIEC菌株是E.coli LF82。AIEC在IBD发生发展中的作用和机制尚未阐明，尤其其是否与机体的某些免疫异常状态存在协同作用共同促进IBD的发病尚不清楚。Toll样受体（Toll-like receptors-5）是能直接识别病原菌分子结构的一类模式识别受体，在机体抵御病原菌入侵的过程中起着重要作用。其中Toll样受体-5（Toll-likereceptors-5，TLR5）可识别细菌鞭毛蛋白并与之结合，激活核因子活化B细胞κ轻链增强子（nuclear factor kappa-light-chain-enhancer of activated B cells，NF-κB），进而刺激大量炎症细胞因子产生，但TLR5参与炎症反应的具体机制还有待进一步研究。本研究旨在明确AIEC菌株E.coli LF82在体外肠上皮屏障及体内的作用，并探讨TLR5在此过程中扮演的角色及可能的分子机制，从而为阐明特殊类型的肠道大肠杆菌在IBD发生、发展中的作用提供理论依据。【方法】 1.利用Caco-2细胞系模型建立体外肠上皮屏障，通过测定跨上皮细胞电阻（transepithelial electrical resistance，，TEER）观察E.coli LF82对屏障完整性的影响，测定荧光黄通过率研究E.coli LF82对肠屏障通透性的影响，通过免疫荧光化学和Western blot观察E.coli LF82对细胞间紧密连接的影响，通过ELISA研究E.coliLF82对细胞培养上清中炎症细胞因子的影响； 2.建立葡聚糖硫酸钠（dextran sulfate sodium，DSS）诱导C57BL/10C小鼠实验性结肠炎模型，造模前一周向实验动物灌胃给予109CFU/天E.coli LF82，通过疾病活动性评价指标、组织学表现、结肠组织MPO活性研究比较E.coli LF82对IBD实验动物的结肠炎症作用，通过透射电镜和免疫荧光化学观察小鼠结肠超微结构和细胞骨架的变化，通过糖原染色、天狼星红染色分别评估小鼠粘液产生能力和纤维化程度，通过Bio-plex、实时定量PCR和Western blot测定血清、组织中炎症细胞因子及炎症反应通路相关分子的变化，通过免疫组织化学和Western blot检测实验小鼠体内TLR5的表达证实TLR5是否参与E.coli LF82的致病过程； 3.利用TLR5基因敲除小鼠探讨TLR5对E.coli LF82作用的影响及可能机制。通过疾病活动性评价指标、组织学表现、超微结构、结肠组织MPO活性、糖原染色、天狼星红染色及血清、组织中炎症细胞因子的测定等比较感染了E.coli LF82的野生型小鼠和TLR5基因敲除小鼠对DSS造模的表现，探讨TLR5在E.coli LF82感染结肠炎小鼠致病过程中的作用。【结果】 1. E.coli LF82可降低肠上皮细胞屏障跨上皮电阻TEER，增加肠上皮细胞屏障对荧光黄大分子的通过率，改变肠上皮细胞屏障紧密连接蛋白的分布和含量，并且影响肠上皮细胞屏障TNF-α等炎症细胞因子的分泌； 2.通过急性活动相关性指标评价发现E.coli LF82可加重DSS诱导小鼠结肠炎症，导致小鼠结肠超微结构和细胞骨架的改变，E.coli LF82还可降低DSS诱导小鼠结肠粘液分泌能力，导致结肠组织纤维化程度增加，同时增加小鼠血清和结肠炎炎症细胞因子的释放，伴随着磷酸化NF-κB P65、P38的表达上调以及TLR5mRNA和蛋白水平的升高； 3.相比野生型小鼠，E.coli LF82对DSS诱导TLR5KO小鼠的结肠炎症程度加重更明显，E.coli LF82可进一步增加DSS诱导TLR5KO小鼠结肠炎炎症细胞因子的分泌释放。经E. coli LF82处理后，TLR5KO小鼠结肠组织磷酸化P38表达升高，但是升高的程度比野生型小鼠要低，而磷酸化NF-κB P65的表达则变化不明显。【结论】我们的研究证实E.coli LF82可破坏体外肠上皮屏障完整性和通透性，并刺激炎症细胞因子分泌；E.coli LF82可加重DSS诱导实验性小鼠结肠炎，细菌鞭毛蛋白/TLR5相互结合并激活MAPK和NF-κB炎症信号通路可能参与介导这种加重作用；在TLR5KO小鼠中，E.coli LF82也可加重DSS小鼠的结肠炎症，表明除了通过TLR5激活的MAPK、NF-κB通路以外，E. coli LF82还可通过其他方式或机制破坏上皮完整，并加重DSS小鼠结肠炎症。
[Abstract]:BACKGROUND & OBJECTIVE :

In recent years , the pathogenesis of inflammatory bowel disease ( IBD ) has been increasing , but the etiology is unknown , the pathogenesis is not clear , and it is difficult to cure completely . The most representative AIEC strain is E . coli LF82 . The role and mechanism of Toll - like receptor - 5 ( TLR5 ) in the development of IBD is not clear .

Methodology

1 . Using Caco - 2 cell line model to establish an in vitro intestinal epithelial barrier , the influence of E . coli LF82 on the barrier integrity was observed by measuring the effect of E . coli LF82 on the permeability of the intestinal barrier . The effect of E . coli LF82 on the intestinal barrier permeability was investigated by immunofluorescence chemistry and Western blot .

2 . The experimental colitis model of C57BL / 10C mice induced by dextran sulfate sodium ( DSS ) was established . 109CFU / day E . coli LF82 was administered to the experimental animals during the previous week . The changes of colonic ultrastructure and cytoskeletal structure were assessed by means of transmission electron microscope and immunofluorescence . The changes of inflammatory cytokines and inflammatory response pathways were assessed by means of transmission electron microscopy and Western blot . The expression of TLR5 in the mice was detected by immunohistochemistry and Western blot .

3 . The effect and possible mechanism of TLR5 on E . coli LF82 were studied by using TLR5 gene knockout mice . The effect of TLR5 in the pathogenesis of E . coli LF82 infected mice was investigated .

The result is not valid .

1 . E . coli LF82 can decrease the trans - cutaneous resistance TEER of intestinal epithelial cell barrier , increase the passage rate of intestinal epithelial cell barrier to fluorescent yellow macromolecule , change the distribution and content of tight junction protein of intestinal epithelial cell barrier , and influence the secretion of inflammatory cytokines such as TNF - 伪 in intestinal epithelial cell barrier .

2 . It was found that E . coli LF82 could aggravate the colonic inflammation of mice induced by DSS , which resulted in the changes of colonic ultrastructure and cytoskeletal structure of mice . E . coli LF82 could also decrease the secretion capacity of colonic mucus of mice induced by DSS , which resulted in the increase of colonic tissue fibrosis , while increasing the release of inflammatory cytokines in serum and colitis of mice , accompanied by up - regulation of phosphorylated NF - 魏B P65 and P38 , and increase of TLR5mRNA and protein level .

3 . Compared with wild type mice , E . coli LF82 increased the colonic inflammation degree of TLR5KO mice induced by DSS . E . coli LF82 could further increase the secretion release of inflammatory cytokines induced by DSS induced TLR5KO mice . After treatment with E . coli LF82 , the expression of phosphorylated P38 in colon tissue of TLR5KO mice increased , but the degree of elevation was lower than that of wild type mice , while the expression of phosphorylated NF - 魏B P65 was not obvious .

Conclusion

Our study confirmed that E . coli LF82 could destroy the integrity and permeability of the intestinal epithelial barrier in vitro and stimulate the secretion of inflammatory cytokines ;
E . coli LF82 may aggravate DSS - induced colitis , bacterial flagellin / TLR5 binding to each other and activate MAPK and NF - 魏B inflammatory signaling pathways may be involved in mediating this emphasis ;
In TLR5KO mice , E . coli LF82 may also aggravate colonic inflammation in DSS mice , suggesting that E . coli LF82 may destroy the epithelium intact by other means or mechanisms in addition to MAPK , NF - 魏B pathways activated by TLR5 , and exacerbate colon inflammation in DSS mice .

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R574

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