芦丁抑制大肠杆菌侵袭鸡肺Ⅱ型上皮细胞的作用机制研究

发布时间：2018-04-13 13:19

本文选题：芦丁 + 鸡肺Ⅱ型上皮细胞　；参考：《吉林大学》2017年硕士论文

【摘要】：鸡大肠杆菌病是由埃希氏大肠杆菌的某些致病菌株引起的鸡的一种细菌性传染病。禽致病性大肠杆菌(APEC)往往会导致鸡鸭等禽类心包炎、肝周炎、气囊炎、脑膜炎、败血症等症状,APEC感染时可通过破坏气-血屏障进入血液循环,最终导致败血症的发生。鸡肺Ⅱ型上皮细胞在维持气-血屏障功能方面具有重要的作用。先前实验室已对鸡肺Ⅱ型上皮细胞进行了分离纯化,通过碱性磷酸酶和电镜观察发现所获得的细胞具有上皮细胞的特点。为了进一步确定鸡肺Ⅱ型上皮细胞的纯度,我们采取流式细胞术和免疫荧光法,通过CD74、TTF1、CK19、CK18、Vimentin等特异抗体染色,来确定细胞纯度。流式细胞术检测发现,鸡肺Ⅱ型上皮细胞占分离细胞数的96%,成纤维细胞占有0.7%,非肺的杂细胞占2%左右。细胞免疫荧光检测发现,除Vimentin外,其他抗体染色均可见特异性的免疫荧光。细胞的高纯度为探讨芦丁抑制大肠杆菌侵袭鸡肺Ⅱ型上皮细胞作用机理奠定了基础。APEC含有许多毒力因子,包括铁摄取相关基因(Iuc D、fyu A),I型菌毛A(Fim A、Fim B、Fim C),卷曲菌毛A(Csg A、Csg B),外膜蛋白A(Omp A)等。这些毒力因子与细菌铁摄取、代谢、黏附和侵袭等密切相关。细菌胞外聚集的信号分子AI-2可以通过群体感应系统对毒力因子转录水平进行调控,影响细菌的致病性。因此,在细菌方面,主要探讨了芦丁对大肠杆菌群体感应系统的影响,结果发现用50μg/m L和25μg/m L芦丁处理APEC-O78 4h时,相关毒力基因(Rpos、H-NS、iuc D,fyu A,Csg A、Csg B、Flic、Lsr B、Lsr K、Luxs、Pfs)m RNA分泌水平显著下降(p0.05),芦丁可以通过降低AI-2的分泌来影响细菌生存压力基因的表达进而干扰群体感应系统,且芦丁可以降低APEC-O78生物膜的形成能力。在细胞方面,采取了转录组测序技术探讨了大肠杆菌侵袭鸡肺Ⅱ型上皮细胞的致病机理和芦丁的干预机制。首先,我们通过涂板法、LDH损伤测定法,确定了样品最佳处理方式为:50μg/m L芦丁预处理细胞4h后,再感染APEC-O78 4h后取样,进行转录组测序。结果发现,鸡肺II型上皮细胞(CP II)感染APEC-O78 4h,通过分析比较感染APEC-O78组和空白组,共有1390个表达差异显著的基因(P0.05),其中上调基因有587个,下调基因有803个。转录组结果测序分析CP II细胞在感染APEC-O78后,免疫应答涉及到NF-kappa B信号通路、细胞凋亡、炎症应答紧密连接、细胞因子—细胞因子受体相互作用、Toll样受体等信号通路。CP II细胞预加药50μg/m L芦丁4h,再除去药物后感染APEC-O78 4h,通过分析比较感染APEC-O78组和加药组,共有222个表达差异显著的基因(P0.05),其中上调基因有59个,下调基因有163个,芦丁可能通过调节鸡肺Ⅱ型上皮细胞的大肠杆菌感染、吞噬体、间隙连接以及糖酵解/糖异生、糖胺聚糖生物合成、磷酸戊糖途径等途径。综上所述,从细菌和细胞两个方面研究了芦丁抑制大肠杆菌侵袭鸡肺Ⅱ型上皮细胞的作用机理。在细菌方面,芦丁可以通过抑制AI-2的分泌而干扰群体感应系统,降低毒力基因的表达,从而抑制大肠杆菌对鸡肺II型上皮细胞的损伤。在细胞方面,转录组学分析显示,芦丁可能通过调节鸡肺Ⅱ型上皮细胞的大肠杆菌感染、吞噬体、间隙连接以及糖酵解等途径,抑制大肠杆菌所造成的损伤。这为芦丁在兽医学临床和其他生物学领域的应用奠定基础。
[Abstract]:Chicken Colibacillosis is a bacterial infectious disease caused by some pathogenic strains of Escherichia coli in chicken. Avian pathogenic Escherichia coli (APEC) often lead to Pope pericarditis, parahepatitis, airsacculitis, meningitis, sepsis and other symptoms, APEC infection can damage the blood air barrier into blood circulation, eventually lead to the occurrence of sepsis. Plays an important role in chicken lung type II epithelial cells in maintaining blood air barrier function. The previous laboratory has purified chicken lung type II epithelial cells by alkaline phosphatase and electron microscope characteristics was obtained by cells with epithelial cells in order to further determine the purity. Chicken type II pulmonary epithelial cells, we use flow cytometry and immunofluorescence method, through CD74, TTF1, CK19, CK18, Vimentin and other specific antibody staining to determine the purity of cells. Flow cytometry. Survey found that chicken lung type II epithelial cells accounted for 96% of the number of isolated cells, fibroblast cells occupy 0.7%, miscellaneous non lung accounted for about 2%. Cell immunofluorescence assay, except Vimentin, other visible immunofluorescence antibody staining specificity. High purity cell of rutin inhibiting Escherichia coli invasion effect of chicken type II epicytes mechanism laid the foundation of.APEC contains many virulence factors, including iron uptake related genes (Iuc D, Fyu A), I A (Fim A, pili Fim B, Fim C), A (Csg A, crimp pili Csg B), outer membrane protein A (Omp A). These virulence factors and bacterial iron uptake, metabolism, adhesion and invasion of closely related bacterial extracellular signal molecules. AI-2 can gather through quorum sensing system on virulence factor transcription regulation, effect of pathogenic bacteria. Therefore, in bacteria, mainly discusses the rutin on Escherichia coli group Effect of system, the results indicated that 50 g/m L and 25 g/m L APEC-O78 rutin 4h, virulence related genes (Rpos, H-NS, IUC D, Fyu A, Csg A, Csg B, Flic, Lsr B, Lsr K, Luxs, Pfs) m RNA secretion level was significantly decreased (P0.05) that rutin can secrete to reduce the effect of AI-2 gene expression of the bacterial survival pressure and interfere with quorum sensing system, forming ability and rutin can reduce APEC-O78 biofilm. In cells, take transcriptome sequencing technology to discuss the intervention mechanism of the pathogenic mechanism of rutin and Escherichia coli invasion of chicken lung type II epithelial cells. First of all, we through the coated board, LDH damage assay, determine the sample is the best way to deal with: 50 g/m L rutin 4H cells after infected the APEC-O78 4h after sampling, by transcriptome sequencing. The results showed that chicken type II alveolar epithelial cells (CP II) APEC-O78 4H infection, through the analysis of the ratio Compared with APEC-O78 infection group and blank group, there were significant differences in the expression of the 1390 genes (P0.05), including 587 up-regulated genes, 803 genes were down regulated. The transcriptome sequencing of CP in II cells after APEC-O78 infection, the immune response involves NF-kappa B signaling pathway, apoptosis, inflammatory response linked closely. Cytokine and cytokine receptor interaction, Toll like receptor signal pathway of.CP II cells pre dosing 50 g/m L 4h APEC-O78 4H and rutin, remove the infection drugs, through the analysis and comparison of APEC-O78 infection group and treatment group, there were significant differences in the expression of the 222 genes (P0.05), including 59 up-regulated genes and 163 genes were down regulated, rutin may regulate the chicken lung type II epithelial cells infected with Escherichia coli, phagosome, gap junction, glycolysis / gluconeogenesis, glycosaminoglycan biosynthesis pathway by way of sugar. In summary, The mechanism from the two aspects of bacteria and cells of rutin inhibiting Escherichia coli invasion of chicken lung type II epithelial cells. In bacteria, rutin can interfere with quorum sensing system by inhibiting the secretion of AI-2, reduce the expression of virulence genes, thereby inhibiting Escherichia coli of chicken type II alveolar epithelial cell injury in the cell., transcriptome analysis showed that rutin may regulate the chicken lung type II epithelial cells infected with Escherichia coli, phagosome, gap junction and glycolytic pathway, inhibition caused by Escherichia coli damage. This lays the foundation for the application of Rutin in veterinary clinical and other fields of biology.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.61

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