本文選題：乳酸菌　切入點(diǎn)：RegIIIγ　出處：《吉林農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：腸道上皮是機(jī)體內(nèi)最大的黏膜屏障,其產(chǎn)生的抑菌因子胰島再生源蛋白IIIγ(regenerating islet-derived protein IIIγ,RegIIIγ)是腸道先天黏膜免疫中重要成分。RegIIIγ為一種可以直接殺菌的C型凝集素,研究發(fā)現(xiàn)髓樣分化因子88(myeloid differentiation factor88,MyD88)及IL-23/IL-22等因子的產(chǎn)生對(duì)RegIIIγ的表達(dá)其促進(jìn)作用。健康腸道內(nèi)RegIIIγ具有較強(qiáng)的殺菌能力,在防止共生菌和致病菌穿透腸道屏障及炎癥性腸病(Inflammatory Bowel Disease,IBD)發(fā)病機(jī)制中發(fā)揮重要作用。乳酸菌作為腸道黏膜表面優(yōu)勢(shì)菌群具有諸多益生功能,可通過(guò)與腸道上皮相互作用調(diào)節(jié)腸道內(nèi)環(huán)境穩(wěn)定及加強(qiáng)免疫防御,從而促進(jìn)腸道健康,同時(shí)在IBD的防治方面也具有一定療效。然而乳酸菌在IBD中的作用機(jī)理仍不明確,尤其在乳酸菌與腸道上皮細(xì)胞相互作用過(guò)程中對(duì)抑菌蛋白R(shí)egIIIγ調(diào)控機(jī)制尚不清晰。本研究通過(guò)分析乳酸菌干預(yù)的小鼠機(jī)體內(nèi)RegIIIγ的量的變化,初步探討RegIIIγ在乳酸菌免疫防御及拮抗腸炎黏膜免疫中的作用及分子機(jī)制。研究?jī)?nèi)容如下:(1)混合乳酸菌對(duì)健康小鼠腸道RegIIIγ的量的影響本實(shí)驗(yàn)利用前期已篩選出的具有較強(qiáng)益生活性的混合乳酸菌干預(yù)健康小鼠,通過(guò)腸道HE染色、熒光定量PCR、免疫熒光及酶聯(lián)免疫吸附試驗(yàn)技術(shù)檢測(cè)干預(yù)前后小鼠腸道組織結(jié)構(gòu)變化、Reg IIIγ及細(xì)胞因子包括MyD88、IL-23(IL-12p40,IL-23p19)、IL-22、IL-17A及TNF-α的量的變化,初步探索在正常腸道內(nèi)乳酸菌對(duì)腸道黏膜先天免疫因子RegIIIγ及細(xì)胞因子調(diào)控的分子機(jī)制。此外,利用滅活菌及菌液培養(yǎng)上清干預(yù)正常小鼠,通過(guò)Q-PCR技術(shù)檢測(cè)干預(yù)前后小鼠腸道內(nèi)RegIIIγmRNA水平的變化,分析混合乳酸菌所分泌的代謝產(chǎn)物對(duì)腸道中RegIIIγ的影響。結(jié)果表明:混合乳酸菌定植正常小鼠腸道后RegIIIγ的量顯著增高,與對(duì)正常照組相比具有顯著性差異。組織學(xué)分析顯示,乳酸菌定植后腸道組織結(jié)構(gòu)正常,無(wú)炎癥產(chǎn)生。且混合乳酸菌干預(yù)14d后,小鼠腸道中MyD88、IL-23及IL-22的量顯著增高;而IL-17A及TNF-α略有升高,但與對(duì)照組無(wú)顯著差異。此外混合乳酸菌干預(yù)的小鼠腸道中,僅活菌干預(yù)組和熱滅活菌干預(yù)組中RegIIIγ的量顯著提高,而乳酸菌培養(yǎng)上清組與對(duì)照組相比無(wú)顯著性差異。(2)混合乳酸菌誘導(dǎo)的Reg IIIγ在細(xì)菌性結(jié)腸炎黏膜免疫中的作用研究本實(shí)驗(yàn)首先利用嚙齒類檸檬酸桿菌(C.rodentium)感染健康小鼠建立腸炎模型,并應(yīng)用不同劑量(1×109cfu及1×1010cfu)混合乳酸菌對(duì)腸炎小鼠進(jìn)行干預(yù)。混合乳酸菌組干預(yù)14d,每天監(jiān)測(cè)各組小鼠體重變化。乳酸菌干預(yù)7d及14d處死小鼠,取出各組結(jié)腸內(nèi)容物及腸段進(jìn)行C.rodentium定植量的測(cè)定及結(jié)腸病理組織學(xué)檢測(cè),同時(shí)應(yīng)用Q-PCR、IF及ELISA檢測(cè)RegIIIγ及細(xì)胞因子MyD88、IL-23(IL-12p40,IL-23p19)、IL-22、IL-17A及TNF-α表達(dá)量的變化,初步分析在C.rodentium所致腸炎中混合乳酸菌的作用。結(jié)果表明,腸炎模型組隨時(shí)間推移機(jī)體體重顯著降低,同時(shí)結(jié)腸中C.rodentium定植量顯著增多,結(jié)腸組織結(jié)構(gòu)嚴(yán)重破壞。而經(jīng)混合乳酸菌干預(yù)后C.rodentium結(jié)腸內(nèi)定植量顯著下調(diào),小鼠體重呈恢復(fù)趨勢(shì),結(jié)腸病變得到顯著改善,其中以1×1010cfu干預(yù)組效果最為顯著。與正常對(duì)照組相比,腸炎模型組小鼠結(jié)腸RegIIIγ及細(xì)胞因子MyD88、IL-23、IL-22、IL-17A及TNF-α顯著上調(diào)并隨著炎癥程度的增高逐漸升高。而混合乳酸菌干預(yù)組與腸炎模型組相比RegIIIγ及細(xì)胞因子MyD88、IL-23、IL-22、IL-17A及TNF-α表達(dá)量顯著降低,并隨著干預(yù)天數(shù)的增加表達(dá)水平進(jìn)一步下調(diào),其中以1×1010cfu劑量干預(yù)組對(duì)RegIIIγ及相關(guān)因子的影響最為顯著。綜上所述,乳酸菌的自身成分或其自身結(jié)構(gòu)而并非分泌的代謝產(chǎn)物可在一定程度上引起MyD88、IL-23及IL-22的升高繼而促進(jìn)RegIIIγ增多。且乳酸菌的定植有助于降低機(jī)體炎性反應(yīng),恢復(fù)機(jī)體免疫平衡以達(dá)到緩解腸炎的作用。進(jìn)一步研究表明乳酸菌緩解IBD的作用機(jī)制可能與降低RegIIIγ異常的表達(dá)有關(guān)。
[Abstract]:Is the largest intestinal mucosal barrier in the body, then the islet inhibitory factor III protein gamma (regenerating islet-derived source protein III y, RegIII y.RegIII y) is an important component of innate mucosal immunity in the gut as a direct bactericidal C type lectin, the research found that myeloid differentiation factor 88 (myeloid differentiation factor88 MyD88, IL-23/IL-22) and cytokines on the expression of RegIII gamma. Its role in promoting the healthy intestine RegIII gamma has strong sterilization ability, in the prevention of commensal and pathogenic bacteria penetrate the intestinal barrier and inflammatory bowel disease (Inflammatory Bowel, Disease, IBD) play an important role in the pathogenesis of intestinal mucosal surface. The lactic acid bacteria as advantage bacteria have many probiotic functions, with intestinal epithelial interactions regulate intestinal homeostasis and enhance the immune defense, so as to promote intestinal health, with In the prevention and treatment of IBD have a certain effect. However, lactic acid bacteria in the IBD mechanism is not clear, especially in the process of lactic acid bacteria and intestinal epithelial cells in the interaction of RegIII inhibitory protein gamma control mechanism is not clear. The changes of mice through analysis of lactic acid bacteria in the intervention of RegIII gamma amount preliminary study of RegIII gamma, lactic acid bacteria in immune defense and antagonistic enteritis mucosal immunity function and mechanism. The contents are as follows: (1) effect of mixed lactic acid bacteria on healthy mice intestinal RegIII gamma quantity has strong activity of probiotic mixed lactic acid bacteria in this experiment using pre screened intervention mice through the intestinal tract HE staining, fluorescence quantitative PCR, immunofluorescence and ELISA structural changes of intestinal tissue in mice were detected before and after intervention test technology, Reg III gamma and cytokines including MyD88, IL-23 (IL-12p40, IL-23p 19), IL-22, IL-17A and TNF- changes in the amount of alpha, preliminary exploration on normal intestinal lactic acid bacteria on intestinal mucosal innate immune factor RegIII gamma and cytokine regulation mechanism. In addition, the supernatant intervention in normal mice using inactivated bacteria and bacteria, through the change of mouse intestinal RegIII gamma mRNA levels before and after the intervention of Q-PCR technology, analysis of the effect of metabolites secreted by the mixture of lactic acid bacteria on intestinal RegIII gamma. The results showed that the mixture of lactic acid bacteria in normal mouse intestine after RegIII gamma were significantly higher compared with the normal control group, with significant difference. Histological analysis showed that the lactic acid bacteria of intestinal structure is normal. No inflammation. And, mixed lactic acid bacteria 14d treated MyD88 mice in the gut, IL-23 and IL-22 were significantly increased; while IL-17A and TNF- alpha was slightly increased, but no significant difference with the control group. In addition to mixed lactic acid bacteria Intestinal intervention, only viable intervention group and heat inactivated bacteria in the intervention group RegIII gamma were significantly increased, and lactic acid bacteria culture supernatant group and the control group had no significant difference. (2) effect of Reg induced by III mixture of lactic acid bacteria in bacterial colitis mucosal immunity in the research the first use of rodent Citrobacter (C.rodentium) infection in healthy mice colitis model, and the application of different doses (1 * 109cfu and 1 * 1010cfu) mixed lactic acid bacteria to intervene enteritis mice. Mixed lactic acid bacteria 14d intervention, body weight changes of mice were monitored daily. The lactic acid bacteria 7d and 14d intervention mice were killed remove the determination and colonic histopathology were detected the contents of the colon and intestinal C.rodentium colonization rate, while application of Q-PCR, detection of MyD88 RegIII gamma and cytokines IF and ELISA, IL-23 (IL-12p40, IL-23p19), IL-22, expression of IL-17A and TNF- alpha The amount of change, a preliminary analysis of mixed lactic acid bacteria in C.rodentium induced enteritis in rats. Results showed that the rats in the model group with the passage of time the body weight decreased significantly, while C.rodentium in colon colonization rate increased significantly, the colon tissue destruction of structure. And the mixed lactic acid bacteria after intervention C.rodentium colon colonization was significantly reduced, the body weight in mice recovery trend, colonic lesions improved significantly, with 1 * 1010cfu group had the best effect. Compared with the normal control group, the rats in the model group colon RegIII mouse gamma and cytokines MyD88, IL-23, IL-22, IL-17A and TNF- significantly increased with the increase of alpha and the degree of inflammation was gradually increased. While the mixed lactic acid intervention group the rats in the model group compared with RegIII gamma and cytokines MyD88, IL-23, IL-22, IL-17A and the expression of TNF- alpha was significantly reduced with the increase in the number of levels of further intervention The cut, with 1 * 1010cfu effect on RegIII gamma dose intervention group and related factors are most significant. In summary, lactic acid bacteria or its components of its structure and metabolites is not secreted can cause MyD88 to a certain extent, the increase in IL-23 and IL-22 and promote RegIII gamma increase. And lactic acid bacteria colonized with helps to reduce the inflammatory response, restore the immune balance to relieve enteritis effect. Further study showed that the lactic acid bacteria relief mechanism of IBD may be related to the decrease of RegIII gamma abnormal expression.

【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.3

【共引文獻(xiàn)】

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