本文選題：蘆丁 + 雞肺Ⅱ型上皮細胞��； 參考：《吉林大學》2017年碩士論文

【摘要】：雞大腸桿菌病是由埃希氏大腸桿菌的某些致病菌株引起的雞的一種細菌性傳染病。禽致病性大腸桿菌(APEC)往往會導致雞鴨等禽類心包炎、肝周炎、氣囊炎、腦膜炎、敗血癥等癥狀,APEC感染時可通過破壞氣-血屏障進入血液循環(huán),最終導致敗血癥的發(fā)生。雞肺Ⅱ型上皮細胞在維持氣-血屏障功能方面具有重要的作用。先前實驗室已對雞肺Ⅱ型上皮細胞進行了分離純化,通過堿性磷酸酶和電鏡觀察發(fā)現(xiàn)所獲得的細胞具有上皮細胞的特點。為了進一步確定雞肺Ⅱ型上皮細胞的純度,我們采取流式細胞術和免疫熒光法,通過CD74、TTF1、CK19、CK18、Vimentin等特異抗體染色,來確定細胞純度。流式細胞術檢測發(fā)現(xiàn),雞肺Ⅱ型上皮細胞占分離細胞數(shù)的96%,成纖維細胞占有0.7%,非肺的雜細胞占2%左右。細胞免疫熒光檢測發(fā)現(xiàn),除Vimentin外,其他抗體染色均可見特異性的免疫熒光。細胞的高純度為探討蘆丁抑制大腸桿菌侵襲雞肺Ⅱ型上皮細胞作用機理奠定了基礎。APEC含有許多毒力因子,包括鐵攝取相關基因(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。這些毒力因子與細菌鐵攝取、代謝、黏附和侵襲等密切相關。細菌胞外聚集的信號分子AI-2可以通過群體感應系統(tǒng)對毒力因子轉錄水平進行調(diào)控,影響細菌的致病性。因此,在細菌方面,主要探討了蘆丁對大腸桿菌群體感應系統(tǒng)的影響,結果發(fā)現(xiàn)用50μg/m L和25μg/m L蘆丁處理APEC-O78 4h時,相關毒力基因(Rpos、H-NS、iuc D,fyu A,Csg A、Csg B、Flic、Lsr B、Lsr K、Luxs、Pfs)m RNA分泌水平顯著下降(p0.05),蘆丁可以通過降低AI-2的分泌來影響細菌生存壓力基因的表達進而干擾群體感應系統(tǒng),且蘆丁可以降低APEC-O78生物膜的形成能力。在細胞方面,采取了轉錄組測序技術探討了大腸桿菌侵襲雞肺Ⅱ型上皮細胞的致病機理和蘆丁的干預機制。首先,我們通過涂板法、LDH損傷測定法,確定了樣品最佳處理方式為:50μg/m L蘆丁預處理細胞4h后,再感染APEC-O78 4h后取樣,進行轉錄組測序。結果發(fā)現(xiàn),雞肺II型上皮細胞(CP II)感染APEC-O78 4h,通過分析比較感染APEC-O78組和空白組,共有1390個表達差異顯著的基因(P0.05),其中上調(diào)基因有587個,下調(diào)基因有803個。轉錄組結果測序分析CP II細胞在感染APEC-O78后,免疫應答涉及到NF-kappa B信號通路、細胞凋亡、炎癥應答緊密連接、細胞因子—細胞因子受體相互作用、Toll樣受體等信號通路。CP II細胞預加藥50μg/m L蘆丁4h,再除去藥物后感染APEC-O78 4h,通過分析比較感染APEC-O78組和加藥組,共有222個表達差異顯著的基因(P0.05),其中上調(diào)基因有59個,下調(diào)基因有163個,蘆丁可能通過調(diào)節(jié)雞肺Ⅱ型上皮細胞的大腸桿菌感染、吞噬體、間隙連接以及糖酵解/糖異生、糖胺聚糖生物合成、磷酸戊糖途徑等途徑。綜上所述,從細菌和細胞兩個方面研究了蘆丁抑制大腸桿菌侵襲雞肺Ⅱ型上皮細胞的作用機理。在細菌方面,蘆丁可以通過抑制AI-2的分泌而干擾群體感應系統(tǒng),降低毒力基因的表達,從而抑制大腸桿菌對雞肺II型上皮細胞的損傷。在細胞方面,轉錄組學分析顯示,蘆丁可能通過調(diào)節(jié)雞肺Ⅱ型上皮細胞的大腸桿菌感染、吞噬體、間隙連接以及糖酵解等途徑,抑制大腸桿菌所造成的損傷。這為蘆丁在獸醫(yī)學臨床和其他生物學領域的應用奠定基礎。
[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.

【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.61

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