本文選題：炎癥性腸病 + 腸道細(xì)菌�。� 參考：《第四軍醫(yī)大學(xué)》2014年博士論文

【摘要】：【背景和目的】 近年來炎癥性腸�。╥nflammatory bowel disease，IBD）的發(fā)病一直呈上升趨勢，但是病因不明，發(fā)病機(jī)制尚不清楚，且難以徹底治愈，嚴(yán)重影響生命健康。一般認(rèn)為感染、環(huán)境等因素觸發(fā)了遺傳易感個體異常的腸道免疫反應(yīng)，引起腸道持續(xù)的炎癥和組織破壞。腸道菌群作為重要的致病因素，直接或間接地參與了包括IBD在內(nèi)的諸多消化系統(tǒng)疾病的發(fā)生發(fā)展。其中黏附侵襲性大腸桿菌（adherent-invasiveEscherichia coli，AIEC）是近年來受到廣泛關(guān)注的一種IBD可能致病菌，最具代表性的AIEC菌株是E.coli LF82。AIEC在IBD發(fā)生發(fā)展中的作用和機(jī)制尚未闡明，尤其其是否與機(jī)體的某些免疫異常狀態(tài)存在協(xié)同作用共同促進(jìn)IBD的發(fā)病尚不清楚。Toll樣受體（Toll-like receptors-5）是能直接識別病原菌分子結(jié)構(gòu)的一類模式識別受體，在機(jī)體抵御病原菌入侵的過程中起著重要作用。其中Toll樣受體-5（Toll-likereceptors-5，TLR5）可識別細(xì)菌鞭毛蛋白并與之結(jié)合，激活核因子活化B細(xì)胞κ輕鏈增強(qiáng)子（nuclear factor kappa-light-chain-enhancer of activated B cells，NF-κB），進(jìn)而刺激大量炎癥細(xì)胞因子產(chǎn)生，但TLR5參與炎癥反應(yīng)的具體機(jī)制還有待進(jìn)一步研究。本研究旨在明確AIEC菌株E.coli LF82在體外腸上皮屏障及體內(nèi)的作用，并探討TLR5在此過程中扮演的角色及可能的分子機(jī)制，從而為闡明特殊類型的腸道大腸桿菌在IBD發(fā)生、發(fā)展中的作用提供理論依據(jù)。 【方法】 1.利用Caco-2細(xì)胞系模型建立體外腸上皮屏障，通過測定跨上皮細(xì)胞電阻（transepithelial electrical resistance，，TEER）觀察E.coli LF82對屏障完整性的影響，測定熒光黃通過率研究E.coli LF82對腸屏障通透性的影響，通過免疫熒光化學(xué)和Western blot觀察E.coli LF82對細(xì)胞間緊密連接的影響，通過ELISA研究E.coliLF82對細(xì)胞培養(yǎng)上清中炎癥細(xì)胞因子的影響； 2.建立葡聚糖硫酸鈉（dextran sulfate sodium，DSS）誘導(dǎo)C57BL/10C小鼠實驗性結(jié)腸炎模型，造模前一周向?qū)嶒瀯游锕辔附o予109CFU/天E.coli LF82，通過疾病活動性評價指標(biāo)、組織學(xué)表現(xiàn)、結(jié)腸組織MPO活性研究比較E.coli LF82對IBD實驗動物的結(jié)腸炎癥作用，通過透射電鏡和免疫熒光化學(xué)觀察小鼠結(jié)腸超微結(jié)構(gòu)和細(xì)胞骨架的變化，通過糖原染色、天狼星紅染色分別評估小鼠粘液產(chǎn)生能力和纖維化程度，通過Bio-plex、實時定量PCR和Western blot測定血清、組織中炎癥細(xì)胞因子及炎癥反應(yīng)通路相關(guān)分子的變化，通過免疫組織化學(xué)和Western blot檢測實驗小鼠體內(nèi)TLR5的表達(dá)證實TLR5是否參與E.coli LF82的致病過程； 3.利用TLR5基因敲除小鼠探討TLR5對E.coli LF82作用的影響及可能機(jī)制。通過疾病活動性評價指標(biāo)、組織學(xué)表現(xiàn)、超微結(jié)構(gòu)、結(jié)腸組織MPO活性、糖原染色、天狼星紅染色及血清、組織中炎癥細(xì)胞因子的測定等比較感染了E.coli LF82的野生型小鼠和TLR5基因敲除小鼠對DSS造模的表現(xiàn)，探討TLR5在E.coli LF82感染結(jié)腸炎小鼠致病過程中的作用。 【結(jié)果】 1. E.coli LF82可降低腸上皮細(xì)胞屏障跨上皮電阻TEER，增加腸上皮細(xì)胞屏障對熒光黃大分子的通過率，改變腸上皮細(xì)胞屏障緊密連接蛋白的分布和含量，并且影響腸上皮細(xì)胞屏障TNF-α等炎癥細(xì)胞因子的分泌； 2.通過急性活動相關(guān)性指標(biāo)評價發(fā)現(xiàn)E.coli LF82可加重DSS誘導(dǎo)小鼠結(jié)腸炎癥，導(dǎo)致小鼠結(jié)腸超微結(jié)構(gòu)和細(xì)胞骨架的改變，E.coli LF82還可降低DSS誘導(dǎo)小鼠結(jié)腸粘液分泌能力，導(dǎo)致結(jié)腸組織纖維化程度增加，同時增加小鼠血清和結(jié)腸炎炎癥細(xì)胞因子的釋放，伴隨著磷酸化NF-κB P65、P38的表達(dá)上調(diào)以及TLR5mRNA和蛋白水平的升高； 3.相比野生型小鼠，E.coli LF82對DSS誘導(dǎo)TLR5KO小鼠的結(jié)腸炎癥程度加重更明顯，E.coli LF82可進(jìn)一步增加DSS誘導(dǎo)TLR5KO小鼠結(jié)腸炎炎癥細(xì)胞因子的分泌釋放。經(jīng)E. coli LF82處理后，TLR5KO小鼠結(jié)腸組織磷酸化P38表達(dá)升高，但是升高的程度比野生型小鼠要低，而磷酸化NF-κB P65的表達(dá)則變化不明顯。 【結(jié)論】 我們的研究證實E.coli LF82可破壞體外腸上皮屏障完整性和通透性，并刺激炎癥細(xì)胞因子分泌；E.coli LF82可加重DSS誘導(dǎo)實驗性小鼠結(jié)腸炎，細(xì)菌鞭毛蛋白/TLR5相互結(jié)合并激活MAPK和NF-κB炎癥信號通路可能參與介導(dǎo)這種加重作用；在TLR5KO小鼠中，E.coli LF82也可加重DSS小鼠的結(jié)腸炎癥，表明除了通過TLR5激活的MAPK、NF-κB通路以外，E. coli LF82還可通過其他方式或機(jī)制破壞上皮完整，并加重DSS小鼠結(jié)腸炎癥。
[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 - 偽 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 .

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R574

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 Lorena Rodríguez-Bores;Gabriela C Fonseca;Marco A Villeda;Jesús K Yamamoto-Furusho;;Novel genetic markers in inflammatory bowel disease[J];World Journal of Gastroenterology;2007年42期

2 Lani Prideaux;Michael A Kamm;Peter De Cruz;Daniel R van Langenberg;Siew C Ng;Iris Dotan;;Inflammatory bowel disease serology in Asia and the West[J];World Journal of Gastroenterology;2013年37期

本文編號：1742173