本文關(guān)鍵詞：牛分枝桿菌和BCG感染對小鼠樹突狀細(xì)胞精氨酸代謝的影響　出處：《華中農(nóng)業(yè)大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 牛分枝桿菌 精氨酸代謝 樹突狀細(xì)胞 一氧化氮 牛結(jié)核 細(xì)胞凋亡

【摘要】：結(jié)核病是一種由結(jié)核分枝桿菌復(fù)合群(Mycobacterium tuberculosis Complex)引起的重要的人畜共患病,包括人和牛在內(nèi)的多種動物均可以感染結(jié)核病。人與動物的結(jié)核病難以根除的一個重要原因在于結(jié)核分枝桿菌的持續(xù)感染問題。一旦結(jié)核分枝桿菌進(jìn)入機(jī)體細(xì)胞后,機(jī)體的免疫系統(tǒng)會對分枝桿菌做出免疫應(yīng)答,早期主要是細(xì)胞免疫,晚期則是以體液免疫為主,但是結(jié)核分枝桿菌有著極其精巧的免疫逃避和潛伏持續(xù)感染機(jī)制如改變自身的代謝方式、干擾吞噬溶酶體的形成、影響巨噬細(xì)胞的活化進(jìn)而影響T細(xì)胞抗原識別、誘導(dǎo)細(xì)胞發(fā)生凋亡等。 為了探尋牛分枝桿菌持續(xù)感染的潛在分子機(jī)制,前期采用小鼠全基因組芯片技術(shù)檢測了牛分枝桿菌強(qiáng)弱菌株感染小鼠樹突狀細(xì)胞后差異基因轉(zhuǎn)錄表達(dá)情況,發(fā)現(xiàn)了36個差異在2倍以上的表達(dá)基因,其中包括與精氨基酸代謝相關(guān)的基因Slc7A2和Slc7A1,精氨酸是細(xì)胞內(nèi)活性氮中間物(RNI)產(chǎn)生的前體物質(zhì),而RNI是抗細(xì)菌感染的重要物質(zhì),是先天性免疫研究的重點(diǎn)關(guān)注對象。 因此,本論文研究牛分枝桿菌強(qiáng)弱菌株感染對樹突狀細(xì)胞精氨酸代謝途徑的調(diào)節(jié),進(jìn)一步揭示NO(活性氮中間物的一種)的產(chǎn)生機(jī)制及其效應(yīng)反應(yīng),以期為闡明牛分枝桿菌持續(xù)感染和免疫逃避機(jī)制提供理論依據(jù)。本研究主要取得以下結(jié)果： (1)感染對樹突狀細(xì)胞NO產(chǎn)生的影響 采用Griess Reagent系統(tǒng)檢測上清中NO的含量,結(jié)果顯示,感染組中的上清NO含量表現(xiàn)增高趨勢,隨著感染時間的增加,NO的含量也在增加。且在感染24h后,BCG感染組要比M.bovis感染組要高,濃度分別為12.57和12.04μM。 (2)感染對陽離子氨基酸轉(zhuǎn)運(yùn)蛋白(CAT)基因和誘導(dǎo)型一氧化氮合酶轉(zhuǎn)錄表達(dá)的影響 采用相對熒光定量PCR方法,針對精氨酸代謝通路中的幾個關(guān)鍵基因進(jìn)行定量。結(jié)果顯示,與未感染組相比,感染組中的Slc7A2基因的mRNA含量均上調(diào),且BCG感染組中均比M.bovis感染組要高(4.263倍,3.14倍),并且外源添加小鼠IFN-y后,與未添加小鼠IFN-y相比,無論是在感染BCG或者是M.bovis隋況下,Slc7A2的mRNA含量均有較大幅度的上升(5.696倍VS 4.263倍、12.247倍VS 3.14倍)。Slc7A1基因的結(jié)果也表現(xiàn)出相似趨勢；與未感染組相比,iNOS的mRNA含量均上調(diào),且感染BCG要比感染M.bovis高(5.328倍、1.247倍)。由于iNOS是受到Th1型細(xì)胞因子的調(diào)控,在添加小鼠IFN-y后,iNOS的mRNA含量均表現(xiàn)出較大幅度的上調(diào),上調(diào)倍數(shù)分別達(dá)到13.362倍和15.345倍。 (3)感染對精氨酸酶基因的轉(zhuǎn)錄表達(dá)的影響 小鼠樹突狀細(xì)胞中檢測到了兩種亞型的精氨酸酶存在。在感染BCG和M.bovis24h后,兩種arginase雖在DC中均有轉(zhuǎn)錄,但arginaseⅠ比arginaseⅡ的1mRNA水平轉(zhuǎn)錄要多；與未感染組相比較,BCG和M. bovis感染組的arginaseⅠ分別達(dá)到29.925倍和9.812倍,而arginaseⅡ只有0.231倍和0.432倍,由此可推測在DC感染BCG和M. bovis過程中,精氨酸酶Ⅰ型占主導(dǎo)優(yōu)勢。 (4)細(xì)胞凋亡檢測 應(yīng)用PI單染法通過流式細(xì)胞儀檢測樹突狀細(xì)胞凋亡發(fā)生比例,結(jié)果發(fā)現(xiàn)(僅BCG感染組),除了Toll樣受體4的抗體處理組外,其他處理組的凋亡發(fā)生比例均要比BCG感染組低。采用PDTC處理的BCG感染組中,樹突狀細(xì)胞的凋亡比例由21.73%下降到12.7%；TLR2抗體處理組下降到16.82%；TLR2抗體和TLR4抗體共同處理組則下降到16.625%；而TLR4抗體處理組則上升到27.05%。由上可見,NO在一定程度上參與了樹突狀細(xì)胞凋亡的發(fā)生。 (5)細(xì)菌胞內(nèi)存活檢測 預(yù)感染24h后,采用平板計(jì)數(shù)的方法對胞內(nèi)細(xì)菌進(jìn)行培養(yǎng)計(jì)數(shù)。BCG感染組胞內(nèi)菌數(shù)量為139500±28723 CFU/孔,而M.bovis感染組為82500±27000 CFU/孔,差異顯著(p=0.027)。但是,當(dāng)添加IFN-γ(100ng/mL)后,BCG感染組則下降到82500±30740 CFU/孔(p=0.0351), M.bovis則下降到31000+1000 CFU/孔(p=0.0234)。當(dāng)外源添加L-NMMA后,胞內(nèi)菌的數(shù)量則上升。因?yàn)長-NMMA抑制了NO的生成,由此提示NO的水平與細(xì)菌的復(fù)制能力存在反比關(guān)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 鹵 28723 CFU / well , while M.tuberculosis infection group decreased to 82500 鹵 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 .

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392

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