本文關(guān)鍵詞： 腸道菌群 流感病毒FM1 益生菌 動(dòng)物模型 TLR7信號(hào)通路　出處：《暨南大學(xué)》2013年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:探討腸道菌群失調(diào)與流感病毒感染的關(guān)系，以及對(duì)小鼠肺部免疫細(xì)胞TLR7信號(hào)通路的影響，為進(jìn)一步研究腸道菌群對(duì)呼吸系統(tǒng)免疫功能及免疫應(yīng)答的影響提供實(shí)驗(yàn)依據(jù)。 方法: 1.流感病毒FM1用雞胚尿囊腔接種法進(jìn)行擴(kuò)增，測(cè)定Balb/c小鼠感染FM1的LD50； 2.建立腸道菌群失調(diào)小鼠感染流感病毒FM1的模型及益生菌恢復(fù)模型。 3.觀察實(shí)驗(yàn)過程中小鼠精神狀態(tài)及體征改變。 4.檢測(cè)各組小鼠肺指數(shù)變化；小鼠回腸盲腸及肺組織光鏡標(biāo)本的制備及HE染色。 5.分別將各組小鼠肺組織勻漿，用實(shí)時(shí)熒光定量PCR法（RT-qPCR）測(cè)定肺部免疫細(xì)胞中TLR7信號(hào)通路中各種信號(hào)蛋白：Toll樣受體7（Toll-like receptor7, TLR7）、髓樣分化因子88（myeolid differentiation factor88, MyD88）、腫瘤壞死因子相關(guān)激酶6(TNFreceptor associated kinase6,TRAF6)、白細(xì)胞介素-1受體相關(guān)激酶(IL-1receptorassociated kinase4,IRAK4)和核因子-B (nuclear factor-B, NF-B) mRNA的表達(dá)。 6.分別將各組小鼠肺組織勻漿，用Western-blot方法檢測(cè)肺部免疫細(xì)胞中TLR7、NF-B蛋白的表達(dá)。 結(jié)果: 1.成功用雞胚尿囊腔接種法擴(kuò)增出流感病毒FM1；血凝試驗(yàn)測(cè)得FM1病毒滴度1:640；根據(jù)預(yù)實(shí)驗(yàn)結(jié)果，測(cè)得Balb/c小鼠感染FM1流感病毒LD50=5×10-5.56。 2.所選用三種抗生素均不同程度造成小鼠腸道菌群失調(diào)，成功建立腸道菌群失調(diào)及益生菌恢復(fù)模型。 3.菌群失調(diào)各組小鼠肺泡、細(xì)支氣管等正常組織結(jié)構(gòu)紊亂、扭曲，結(jié)締組織增生，肺部血管內(nèi)充血嚴(yán)重，肺間質(zhì)中可見大量中性粒細(xì)胞浸潤。益生菌組小鼠肺部病變明顯減輕，肺組織形態(tài)結(jié)構(gòu)較為完整，中性粒細(xì)胞浸潤數(shù)量減少，腔內(nèi)無滲出。 4.抗生素處理各組小鼠盲腸腫大明顯，腸黏膜上皮水腫、甚至脫落，腸間質(zhì)有輕度充血。益生菌治療組盲腸腫大及腸絨毛脫落癥狀明顯減輕。 5.用流感病毒FM1感染小鼠后，肺勻漿中IL-4和IL-10水平降低，IFN-γ和IL-17水平顯著升高，抗生素破壞正常菌群后再感染流感病毒的小鼠，肺勻漿中IL-4，IL-10水平較單純病毒感染小鼠升高，IFN-γ和IL-17水平降低；給予益生菌治療4d后，可降低肺勻漿中IL-4，IL-10水平，同時(shí)升高IFN-γ和IL-17水平。 6. RT-qPCR實(shí)驗(yàn)證明流感病毒感染可上調(diào)TLR7信號(hào)通路各信號(hào)蛋白mRNA的表達(dá)。菌群失調(diào)可顯著下調(diào)TLR7信號(hào)通路的表達(dá)，尤以TLR-7及NF-κB明顯。使用益生菌治療后，TLR7信號(hào)通路相關(guān)蛋白mRNA的表達(dá)上調(diào)，與菌群失調(diào)組相比，差異及其顯著。 7. Western-blot實(shí)驗(yàn)進(jìn)一步證明病毒感染可致TLR7、NF-B蛋白表達(dá)增高，，腸道菌群失調(diào)可下調(diào)TLR7、NF-B蛋白表達(dá)，益生菌治療后，TLR7、NF-κB蛋白的表達(dá)上調(diào)。 結(jié)論: 流感病毒感染可致小鼠肺組織免疫細(xì)胞中TLR7信號(hào)傳導(dǎo)通路活化，腸道菌群失調(diào)會(huì)抑制TLR7信號(hào)通路活化，益生菌治療后可以通過上調(diào)TLR7信號(hào)傳導(dǎo)通路而發(fā)揮抗流感病毒的作用。
[Abstract]:Objective: to investigate the relationship between intestinal flora imbalance and influenza virus infection and the effect on TLR7 signaling pathway of mouse lung immune cells, and to provide experimental evidence for further study of the effects of intestinal flora on respiratory system immune function and immune response. Methods:. 1. Influenza virus FM1 was amplified by chicken embryo allantoic cavity inoculation. LD50 of Balb/c mice infected with FM1 was determined. 2. Establish the model of influenza virus FM1 infection and probiotic recovery model in mice with intestinal dysbacteriosis. 3. Observe the changes of mental state and signs in mice during the experiment. 4. The changes of lung index in each group, the preparation of light microscopic specimen of ileum caecum and lung tissue and HE staining were detected. 5. the lung tissue homogenate of each group of mice, Real-time fluorescence quantitative PCR assay for the detection of various signal proteins in the TLR7 signaling pathway in lung immune cells: Toll-like receptor 7 Toll-like receptor 7, TLR7, myeloid differentiation factor 88myeolid differentiation factor88, MyD88A, tumor necrosis factor-associated kinase 6 TNFreceptor associated kinase6TRAF6, interleukin-1 receptor phase. The expression of IL-1 receptor-associated kinase4 (IRAK4) and nuclear factor-B (NF-B) mRNA. 6. The lung homogenate of each group of mice was used to detect the expression of TLR7 NF-B protein in lung immune cells by Western-blot method. Results:. 1. The influenza virus FM1 was successfully amplified by chicken embryo allantoic cavity inoculation, the titer of FM1 virus was detected by hemagglutination test 1: 640.The Balb/c mice were infected with FM1 influenza virus LD50=5 脳 10-5.56 according to the results of pre-test. 2. The three antibiotics were used to induce intestinal flora imbalance in mice, and the model of intestinal flora imbalance and probiotic recovery was established successfully. 3. The normal structures of alveoli and bronchioles in mice with dysbacteriosis were disorder, distortion, connective tissue proliferation, pulmonary vascular congestion and neutrophil infiltration in pulmonary interstitium. The morphology and structure of lung tissue were relatively complete, the number of neutrophil infiltration was reduced, and there was no exudation in the lumen. 4. The caecum swelling was obvious, intestinal mucosal epithelium edema, even exfoliation, intestinal interstitial hyperemia, and the symptoms of caecum enlargement and villi exfoliation were obviously alleviated in the probiotics treatment group. 5. The levels of IL-4 and IL-10 in the lung homogenate of mice infected with influenza virus FM1 decreased significantly, and the levels of IFN- 緯 and IL-17 increased significantly. The levels of IL-4n- 緯 and IL-17 in lung homogenate were lower than those in mice infected with simplex virus, and the levels of IL-4- 緯 and IL-17 in lung homogenate were decreased after treatment with probiotics for 4 days, and the levels of IFN- 緯 and IL-17 were also increased after treatment with probiotics for 4 days. 6. RT-qPCR assay showed that influenza virus infection could up-regulate the expression of mRNA in TLR7 signaling pathway, and the imbalance of bacterial flora could significantly down-regulate the expression of TLR7 signaling pathway. Especially TLR-7 and NF- 魏 B, the expression of TLR7 signal pathway related protein mRNA was up-regulated after treatment with probiotics, and the difference was significant compared with that of the dysbacteriosis group. 7. Western-blot assay further demonstrated that the expression of TLR7nF-B protein was increased by virus infection, the expression of TLR7nF-B protein was down-regulated by intestinal flora imbalance, and the expression of TLR7NF-B protein was up-regulated after probiotics treatment. Conclusion:. Influenza virus infection could induce the activation of TLR7 signal transduction pathway in mouse lung immune cells, and intestinal flora imbalance could inhibit the activation of TLR7 signaling pathway. Probiotics treatment could play the role of anti-influenza virus by upregulating TLR7 signal transduction pathway.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R373.13

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 鄒英;夏培元;張杰;;1668例住院患者抗生素使用分析[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2006年07期

2 李秀真,朱萬孚;乳酸桿菌抗腫瘤作用研究進(jìn)展[J];國外醫(yī)學(xué)(腫瘤學(xué)分冊(cè));2004年04期

3 閆小毅;TLR7介導(dǎo)抗病毒免疫應(yīng)答研究進(jìn)展[J];國外醫(yī)學(xué)(免疫學(xué)分冊(cè));2005年05期

4 王菲;TLR家族分子信號(hào)轉(zhuǎn)導(dǎo)新進(jìn)展[J];國外醫(yī)學(xué)(免疫學(xué)分冊(cè));2005年05期

5 趙東;徐桂芳;鄒曉平;;益生菌的作用機(jī)制[J];國際消化病雜志;2012年02期

6 余小萍;傅慧婷;;中醫(yī)藥防治流感的免疫調(diào)節(jié)機(jī)制的研究概況[J];遼寧中醫(yī)雜志;2006年08期

7 南淑玲;潘杰;章健;趙黎;方向明;;升降散對(duì)流感病毒FM1感染小鼠血清IL-1β、IL-10含量的影響[J];遼寧中醫(yī)藥大學(xué)學(xué)報(bào);2011年01期

8 李琛;李充璧;;益生菌的生理作用及特性[J];內(nèi)蒙古醫(yī)學(xué)雜志;2009年06期

9 史立敏;張?jiān)撇?董堅(jiān);柳愛華;陳明清;寶福凱;;IL-17及其與疾病關(guān)系研究進(jìn)展[J];中國熱帶醫(yī)學(xué);2012年01期

10 程秀芳;劉虹;張志焱;谷巍;;兩株益生菌的抑菌性及其合生元對(duì)試驗(yàn)小鼠腸道菌群作用研究[J];齊魯藥事;2010年04期

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