肠道菌群失调对流感病毒FM1感染小鼠肺部免疫细胞TLR7信号通路的影响

发布时间：2018-02-26 14:45

本文关键词： 肠道菌群流感病毒FM1 益生菌动物模型 TLR7信号通路　出处：《暨南大学》2013年硕士论文　论文类型：学位论文

【摘要】：目的:探讨肠道菌群失调与流感病毒感染的关系，以及对小鼠肺部免疫细胞TLR7信号通路的影响，为进一步研究肠道菌群对呼吸系统免疫功能及免疫应答的影响提供实验依据。方法: 1.流感病毒FM1用鸡胚尿囊腔接种法进行扩增，测定Balb/c小鼠感染FM1的LD50； 2.建立肠道菌群失调小鼠感染流感病毒FM1的模型及益生菌恢复模型。 3.观察实验过程中小鼠精神状态及体征改变。 4.检测各组小鼠肺指数变化；小鼠回肠盲肠及肺组织光镜标本的制备及HE染色。 5.分别将各组小鼠肺组织匀浆，用实时荧光定量PCR法（RT-qPCR）测定肺部免疫细胞中TLR7信号通路中各种信号蛋白：Toll样受体7（Toll-like receptor7, TLR7）、髓样分化因子88（myeolid differentiation factor88, MyD88）、肿瘤坏死因子相关激酶6(TNFreceptor associated kinase6,TRAF6)、白细胞介素-1受体相关激酶(IL-1receptorassociated kinase4,IRAK4)和核因子-B (nuclear factor-B, NF-B) mRNA的表达。 6.分别将各组小鼠肺组织匀浆，用Western-blot方法检测肺部免疫细胞中TLR7、NF-B蛋白的表达。结果: 1.成功用鸡胚尿囊腔接种法扩增出流感病毒FM1；血凝试验测得FM1病毒滴度1:640；根据预实验结果，测得Balb/c小鼠感染FM1流感病毒LD50=5×10-5.56。 2.所选用三种抗生素均不同程度造成小鼠肠道菌群失调，成功建立肠道菌群失调及益生菌恢复模型。 3.菌群失调各组小鼠肺泡、细支气管等正常组织结构紊乱、扭曲，结缔组织增生，肺部血管内充血严重，肺间质中可见大量中性粒细胞浸润。益生菌组小鼠肺部病变明显减轻，肺组织形态结构较为完整，中性粒细胞浸润数量减少，腔内无渗出。 4.抗生素处理各组小鼠盲肠肿大明显，肠黏膜上皮水肿、甚至脱落，肠间质有轻度充血。益生菌治疗组盲肠肿大及肠绒毛脱落症状明显减轻。 5.用流感病毒FM1感染小鼠后，肺匀浆中IL-4和IL-10水平降低，IFN-γ和IL-17水平显著升高，抗生素破坏正常菌群后再感染流感病毒的小鼠，肺匀浆中IL-4，IL-10水平较单纯病毒感染小鼠升高，IFN-γ和IL-17水平降低；给予益生菌治疗4d后，可降低肺匀浆中IL-4，IL-10水平，同时升高IFN-γ和IL-17水平。 6. RT-qPCR实验证明流感病毒感染可上调TLR7信号通路各信号蛋白mRNA的表达。菌群失调可显著下调TLR7信号通路的表达，尤以TLR-7及NF-κB明显。使用益生菌治疗后，TLR7信号通路相关蛋白mRNA的表达上调，与菌群失调组相比，差异及其显著。 7. Western-blot实验进一步证明病毒感染可致TLR7、NF-B蛋白表达增高，，肠道菌群失调可下调TLR7、NF-B蛋白表达，益生菌治疗后，TLR7、NF-κB蛋白的表达上调。结论: 流感病毒感染可致小鼠肺组织免疫细胞中TLR7信号传导通路活化，肠道菌群失调会抑制TLR7信号通路活化，益生菌治疗后可以通过上调TLR7信号传导通路而发挥抗流感病毒的作用。
[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- 纬 and IL-17 increased significantly. The levels of IL-4n- 纬 and IL-17 in lung homogenate were lower than those in mice infected with simplex virus, and the levels of IL-4- 纬 and IL-17 in lung homogenate were decreased after treatment with probiotics for 4 days, and the levels of IFN- 纬 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.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R373.13

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