本文關(guān)鍵詞： miR-181a 潰瘍性結(jié)腸炎 TNF-α IκB　出處：《中國人民解放軍醫(yī)學(xué)院》2014年博士論文　論文類型：學(xué)位論文

【摘要】：背景 潰瘍性結(jié)腸炎（ulcerative colitis，UC）主要表現(xiàn)為粘液膿血便、反復(fù)發(fā)作性腹瀉、腹痛，病變主要位于結(jié)腸的粘膜層和粘膜下層，是一種慢性非特異性的結(jié)腸炎癥，屬于炎癥性腸病（inflammatory bowel disease, IBD）的一種。本病的確切病因不明，多種因素如遺傳、感染、環(huán)境、免疫等在其發(fā)病過程中起到了重要的作用。腸道免疫系統(tǒng)調(diào)節(jié)異常，促炎細胞因子分泌增多可能是本病的主要發(fā)病機制，因此探討腸道組織中炎癥相關(guān)的信號轉(zhuǎn)導(dǎo)過程有助于闡明UC的發(fā)病機制。 MicroRNA(miRNA)是一類長度為18-24個核苷酸的內(nèi)源性非編碼單鏈小分子RNA（nocoding RNA, ncRNA）。miRNA通過與靶基因3'非翻譯區(qū)(3'UTR)的完全或不完全配對，降解靶基因mRNA或抑制其翻譯負向調(diào)控基因表達，影響組織和細胞的功能。miRNA廣泛參與生命過程中一系列重要進程，如腫瘤、炎癥、細胞增殖、凋亡、組織器官的形成等。本研究旨在探討UC中miRNA的差異表達以及miRNA-181a通過調(diào)控其靶基因TNF-α在UC發(fā)病機制中的作用，為闡明UC的發(fā)病機制提供新的理論依據(jù)。 目的 對課題小組前期的miRNA芯片結(jié)果進行分析，，選取在UC患者結(jié)腸粘膜中表達差異的5種miRNA：miR-181a、miR-181b、miR-182、miR-146a、miR-101，應(yīng)用實時熒光定量RT-PCR方法進行驗證。并探討miR-181a與其靶基因TNF-α在UC患者腸道粘膜炎癥發(fā)展中的作用及機制，為研究UC的發(fā)病機制提供新的切入點。 方法 1.分析前期miRNA芯片結(jié)果篩選出5種在UC患者結(jié)腸粘膜組織中差異表達的miRNA:miR-181a、miR-181b、miR-182、miR-146a、miR-101，應(yīng)用實時熒光定量RT-PCR法進一步進行驗證。 2.選取miR-181a作為進一步的研究對象，利用生物信息學(xué)方法預(yù)測miR-181a可能的靶基因為TNF-α。Westernblot法檢測UC患者和健康對照組結(jié)腸粘膜中TNF-α的表達變化。 3構(gòu)建TNF-α3’UTR野生型及突變型載體，應(yīng)用雙熒光素酶報告基因?qū)嶒灆z測miR-181a與TNF-α之間的靶向調(diào)控關(guān)系；過表達和抑制表達miR-181a進一步檢測TNF-α在蛋白水平的變化。 4.過表達和抑制表達miR-181a檢測TNF-α及下游IκB的表達變化。 結(jié)果 1.實時熒光定量RT-PCR法檢測發(fā)現(xiàn)在UC患者結(jié)腸粘膜中miR-146a、miR-101表達上調(diào)，miR-181a、miR-181b、miR-182表達下調(diào)。 2. Western blot法顯示UC組結(jié)腸組織中TNF-α蛋白水平的明顯高于健康對照組。 3.熒光素酶基因報告實驗顯示miR-181a可顯著降低TNF-α3’UTR野生型組的熒光表達，而轉(zhuǎn)染TNF-α3’UTR突變體組熒光表達升高；Western blot法顯示過表達miR-181a組TNF-α蛋白水平的表達明顯降低。 4Western blot法顯示過表達miR-181a后TNF-α表達降低而IκB表達升高；抑制表達miR-181a后TNF-α表達升高及IκB表達降低。 結(jié)論 本課題對前期UC的miRNA芯片結(jié)果進行了分析，并應(yīng)用實時熒光定量RT-PCR方法對其中5種miRNA進行檢測，在UC患者結(jié)腸粘膜中miR-146a、miR-101表達上調(diào)；miR-181a、miR-181b、miR-182表達下調(diào)。證實TNF-α是miR-181a直接調(diào)控的靶基因，并可能通過調(diào)節(jié)NF-κB信號轉(zhuǎn)導(dǎo)途徑參與了UC結(jié)腸粘膜炎癥的發(fā)生發(fā)展，為進一步深入研究UC的發(fā)病機制提供了新的途徑。
[Abstract]:background
Ulcerative colitis (ulcerative colitis UC) is mainly mucusbloodandpus, recurrent diarrhea, abdominal pain, lesions are mainly located in the colon mucosa and submucosa, is a kind of chronic nonspecific colitis, belong to inflammatory bowel disease (inflammatory bowel, disease, IBD) is a kind of the exact cause of the disease. Unknown, many factors such as heredity, environment, infection, immunity plays an important role in the disease. The abnormal regulation of intestinal immune system, proinflammatory cytokine secretion may be the main pathogenesis of the disease, so the pathogenesis of inflammation related signal transduction in intestinal tissue contributes to the elucidation of UC.
MicroRNA (miRNA) is a class of endogenous non length of 18-24 nucleotides encoding small single stranded RNA (nocoding RNA ncRNA).MiRNA with target gene 3'untranslated region (3'UTR) of the complete or incomplete pairing, mRNA target gene degradation or inhibit translation negatively regulate gene expression, function and effect.MiRNA cells widely participate in the process of life in a series of important processes, such as tumor, inflammation, cell proliferation, apoptosis, tissue and organ formation. This study aimed to investigate the expression of UC in miRNA and miRNA-181a through the regulation of its target gene TNF- in the pathogenesis of UC and provide a new theoretical basis for elucidating the pathogenesis UC.
objective
The miRNA chip on the topic groupearlier period by analyzing the results of the selected expression of 5 miRNA:miR-181a, the differences in colonic mucosa of UC patients in miR-181b, miR-182, miR-146a, miR-101, by real-time quantitative RT-PCR. Verify and investigate in UC patients of intestinal mucosal inflammation in the development of the role and mechanism of miR-181a and its target gene TNF-, provide a new starting point for studying the pathogenesis of UC.
Method
1. analyze the previous miRNA chip results, and select 5 miRNA:miR-181a, miR-181b, miR-182, miR-146a and miR-101 expressed in colonic mucosa tissue of UC patients. The real-time fluorescent quantitative RT-PCR method is used for further validation.
2. select miR-181a as a further research object, and predict the possible target of miR-181a by bioinformatics. TNF- alpha.Westernblot method is used to detect the expression of TNF- alpha in colonic mucosa of UC patients and healthy controls.
3, we constructed the TNF- alpha 3 'UTR wild type and mutant vector. We used dual luciferase reporter gene assay to detect the target regulatory relationship between miR-181a and TNF- alpha, overexpressed and suppressed expression of miR-181a, and further detected the change of TNF- alpha at protein level.
4. over expression and inhibition of expression of miR-181a detected the expression of TNF- alpha and the expression of I kappa B downstream.
Result
1. the real-time fluorescence quantitative RT-PCR assay found that the expression of miR-146a and miR-101 in the colon mucosa of UC patients was up-regulated, and the expression of miR-181a, miR-181b, and miR-182 was down regulated.
The 2. Western blot method showed that the level of TNF- alpha protein in the colon tissue of UC group was significantly higher than that in the healthy control group.
3. luciferase gene reporter test showed that miR-181a significantly decreased the fluorescence expression of TNF- 3 'UTR wild type group, while the fluorescence expression of TNF- alpha 3' UTR mutant group increased. Western blot showed that the expression of TNF- alpha protein in miR-181a group was significantly lower than that in the miR-181a group.
4Western blot showed that the expression of TNF- alpha was decreased and the expression of I kappa B increased after overexpression of miR-181a, and the expression of TNF- A and the expression of I kappa B decreased after inhibiting the expression of miR-181a.
conclusion
The miRNA chip on the early results of UC were analyzed, and the 5 kinds of miRNA were detected by real-time fluorescence quantitative RT-PCR method, miR-146a in colonic mucosa of UC patients, expression of miR-181a, miR-181b, miR-101; expression of miR-182 alpha confirmed that TNF- was a target gene of miR-181a regulation, and may occur through development regulation of NF- B signal transduction pathway in UC colon inflammation, provides a new way for further research on the pathogenesis of UC.

【學(xué)位授予單位】：中國人民解放軍醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R574.62

【參考文獻】

相關(guān)期刊論文 前1條

1 Jannie Pedersen;Mehmet Coskun;Christoffer Soendergaard;Mohammad Salem;Ole Haagen Nielsen;;Inflammatory pathways of importance for management of inflammatory bowel disease[J];World Journal of Gastroenterology;2014年01期

本文編號：1518252