牛分枝杆菌和BCG感染对小鼠树突状细胞精氨酸代谢的影响

发布时间：2018-01-08 05:05

本文关键词：牛分枝杆菌和BCG感染对小鼠树突状细胞精氨酸代谢的影响　出处：《华中农业大学》2011年硕士论文　论文类型：学位论文

【摘要】：结核病是一种由结核分枝杆菌复合群(Mycobacterium tuberculosis Complex)引起的重要的人畜共患病,包括人和牛在内的多种动物均可以感染结核病。人与动物的结核病难以根除的一个重要原因在于结核分枝杆菌的持续感染问题。一旦结核分枝杆菌进入机体细胞后,机体的免疫系统会对分枝杆菌做出免疫应答,早期主要是细胞免疫,晚期则是以体液免疫为主,但是结核分枝杆菌有着极其精巧的免疫逃避和潜伏持续感染机制如改变自身的代谢方式、干扰吞噬溶酶体的形成、影响巨噬细胞的活化进而影响T细胞抗原识别、诱导细胞发生凋亡等。为了探寻牛分枝杆菌持续感染的潜在分子机制,前期采用小鼠全基因组芯片技术检测了牛分枝杆菌强弱菌株感染小鼠树突状细胞后差异基因转录表达情况,发现了36个差异在2倍以上的表达基因,其中包括与精氨基酸代谢相关的基因Slc7A2和Slc7A1,精氨酸是细胞内活性氮中间物(RNI)产生的前体物质,而RNI是抗细菌感染的重要物质,是先天性免疫研究的重点关注对象。因此,本论文研究牛分枝杆菌强弱菌株感染对树突状细胞精氨酸代谢途径的调节,进一步揭示NO(活性氮中间物的一种)的产生机制及其效应反应,以期为阐明牛分枝杆菌持续感染和免疫逃避机制提供理论依据。本研究主要取得以下结果： (1)感染对树突状细胞NO产生的影响采用Griess Reagent系统检测上清中NO的含量,结果显示,感染组中的上清NO含量表现增高趋势,随着感染时间的增加,NO的含量也在增加。且在感染24h后,BCG感染组要比M.bovis感染组要高,浓度分别为12.57和12.04μM。 (2)感染对阳离子氨基酸转运蛋白(CAT)基因和诱导型一氧化氮合酶转录表达的影响采用相对荧光定量PCR方法,针对精氨酸代谢通路中的几个关键基因进行定量。结果显示,与未感染组相比,感染组中的Slc7A2基因的mRNA含量均上调,且BCG感染组中均比M.bovis感染组要高(4.263倍,3.14倍),并且外源添加小鼠IFN-y后,与未添加小鼠IFN-y相比,无论是在感染BCG或者是M.bovis隋况下,Slc7A2的mRNA含量均有较大幅度的上升(5.696倍VS 4.263倍、12.247倍VS 3.14倍)。Slc7A1基因的结果也表现出相似趋势；与未感染组相比,iNOS的mRNA含量均上调,且感染BCG要比感染M.bovis高(5.328倍、1.247倍)。由于iNOS是受到Th1型细胞因子的调控,在添加小鼠IFN-y后,iNOS的mRNA含量均表现出较大幅度的上调,上调倍数分别达到13.362倍和15.345倍。 (3)感染对精氨酸酶基因的转录表达的影响小鼠树突状细胞中检测到了两种亚型的精氨酸酶存在。在感染BCG和M.bovis24h后,两种arginase虽在DC中均有转录,但arginaseⅠ比arginaseⅡ的1mRNA水平转录要多；与未感染组相比较,BCG和M. bovis感染组的arginaseⅠ分别达到29.925倍和9.812倍,而arginaseⅡ只有0.231倍和0.432倍,由此可推测在DC感染BCG和M. bovis过程中,精氨酸酶Ⅰ型占主导优势。 (4)细胞凋亡检测应用PI单染法通过流式细胞仪检测树突状细胞凋亡发生比例,结果发现(仅BCG感染组),除了Toll样受体4的抗体处理组外,其他处理组的凋亡发生比例均要比BCG感染组低。采用PDTC处理的BCG感染组中,树突状细胞的凋亡比例由21.73%下降到12.7%；TLR2抗体处理组下降到16.82%；TLR2抗体和TLR4抗体共同处理组则下降到16.625%；而TLR4抗体处理组则上升到27.05%。由上可见,NO在一定程度上参与了树突状细胞凋亡的发生。 (5)细菌胞内存活检测预感染24h后,采用平板计数的方法对胞内细菌进行培养计数。BCG感染组胞内菌数量为139500±28723 CFU/孔,而M.bovis感染组为82500±27000 CFU/孔,差异显著(p=0.027)。但是,当添加IFN-γ(100ng/mL)后,BCG感染组则下降到82500±30740 CFU/孔(p=0.0351), M.bovis则下降到31000+1000 CFU/孔(p=0.0234)。当外源添加L-NMMA后,胞内菌的数量则上升。因为L-NMMA抑制了NO的生成,由此提示NO的水平与细菌的复制能力存在反比关系。
[Abstract]:Tuberculosis is an important human and livestock co - infection caused by Mycobacterium tuberculosis complex . One of the most important causes of tuberculosis is tuberculosis . One of the most important reasons for tuberculosis infection is the persistent infection of M.tuberculosis . Once Mycobacterium tuberculosis enters the organism cells , the immune system of the organism will immune response to Mycobacterium tuberculosis . In the early stage , the immune system of the organism will immune response to Mycobacterium tuberculosis . However , Mycobacterium tuberculosis has extremely delicate immune evasion and latent persistent infection mechanism , such as changing its metabolism , interfering with the formation of lysosome , affecting the activation of macrophages , affecting the antigen recognition of T cells , inducing apoptosis , etc . In order to explore the potential molecular mechanism of the persistent infection of Mycobacterium tuberculosis , the expression of differential gene in mouse dendritic cells infected with virulent strain of Mycobacterium tuberculosis was detected in the early stage by using the mouse full genome chip technique . The expression genes related to the metabolism of refined amino acids were found , including the genes Slc7A2 and Slc7A1 related to the metabolism of refined amino acids . The arginine was the precursor substance produced by the active nitrogen intermediate ( RNI ) in the cell , and RNI was an important substance for the anti - bacterial infection , and it was the focus of the research on innate immunity . Therefore , this paper studies the regulation of the pathway of arginine metabolism of dendritic cells by the infection of the virulent strains of Mycobacterium , and further reveals the mechanism and the effect of NO ( one of active nitrogen intermediates ) , with a view to providing a theoretical basis for the clarification of the mechanism of persistent infection and immune evasion of bovine mycobacterium . The study mainly takes the following results : ( 1 ) Effect of Infection on NO Production in Dendritic Cells The content of NO in the supernatant was detected by the Griess Reagent system . The results showed that the content of NO in the infected group increased , and the content of NO increased with the increase of infection time . After 24 h infection , the concentration of NO in BCG was 12.57 and 12.04 渭M , respectively . ( 2 ) Effects of Infection on the Expression of Cationic Amino Acid Transport Protein ( CAT ) Gene and inducible Nitric Oxide Synthase Compared with the uninfected group , the mRNA content of Slc7A2 gene in the infected group was higher than that in the non - infected group ( 4.263 times , 3.14 times ) , and the mRNA content of Slc7A2 increased significantly ( 5.696 times VS 4.263 times , 12.247 times VS 3.14 times ) compared with the non - infected group . The results of the Slc7A1 gene showed similar trends . Compared with the uninfected group , the mRNA content of iNOS was up - regulated , and the infection BCG was higher than that of the infected group ( 5.328 times , 1.247 times ) . Since iNOS was regulated by Th1 type cytokines , the mRNA content of iNOS was up - regulated after the addition of IFN - y to mice , up - regulated by 13.362 - fold and 15.345 - fold , respectively . ( 3 ) Effect of infection on the transcriptional expression of the gene of the enzyme In the presence of BCG and M.bovisat 24 h , the two isoforms were transcribed in both DC and BCG and M.bovisat 24 h , but not only 0.231 and 0.432 times as compared with the uninfected group . ( 4 ) Apoptosis detection In the BCG - infected group treated with PDTC , the percentage of apoptosis of dendritic cells decreased from 21.73 % to 12.7 % , while TLR2 antibody - treated group decreased to 16.82 % . ( 5 ) Bacterial intracellular survival detection After 24 hours of pre - infection , the intracellular bacteria were cultured and counted by plate counting method . The number of intracellular bacteria in BCG - infected group was 139 500 卤 28723 CFU / well , while M.tuberculosis infection group decreased to 82500 卤 30740 CFU / well ( p = 0.0234 ) . However , when L - NMMA was added , the number of intracellular bacteria increased . Because L - NMMA inhibited NO production , it was suggested that the level of NO was inversely related to the replication ability of bacteria .

【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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