本文選題：傳染性法氏囊病毒 + 線粒體　； 參考：《河南科技大學》2017年碩士論文

【摘要】：雞傳染性法氏囊病(Infectious bursal disease,IBD)是由傳染性法氏囊病病毒(Infections bursal diseas virus,IBDV)引起的雞的一種急性、高度接觸性傳染病。IBDV在入侵宿主后迅速復制致使B淋巴細胞大量壞死和凋亡,導致雞的免疫功能受到嚴重損傷,造成被感染家禽嚴重的免疫抑制。傳染性法氏囊病的發(fā)生過程,是宿主與病原多層面作用的復雜過程,目前IBD致病的分子機制并不十分清楚。本研究擬以IBDV感染DT40細胞和非免疫雞胚,通過對線粒體代謝動力學和能量水平的檢測,從代謝層面上揭示IBDV感染細胞的線粒體生物氧化與致病性的關系;通過表達IBDV-VP4蛋白對脂酰CoA轉運的影響,從分子層面探討VP4蛋白分子擬態(tài)對線粒體呼吸鏈能量代謝的影響。1、IBDV感染對B淋巴細胞線粒體能量代謝的影響為研究IBDV感染對宿主免疫細胞線粒體代謝的影響,本實驗以IBDV分離株感染DT40細胞,分別于病毒感染后24 h、48 h、72 h和96 h取樣,細胞計數(shù)并分離細胞線粒體,通過檢測線粒體蛋白含量,線粒體膜電位,肉堿脂酰轉移酶和NADH-CoQ還原酶活性,ATP水平,線粒體脂酰-CoA和NADH、NAD+的含量及NADH/NAD+比值,初步評估IBDV感染對宿主細胞線粒體生物氧化的影響。結果表明,IBDV感染DT40細胞48 h后,線粒體蛋白含量顯著降低(P0.01),膜電位下降,肉堿脂酰轉移酶和NADH-CoQ還原酶活性在感染后24 h開始下降(P0.05),48 h后顯著降低(P0.01);脂酰-CoA和ATP含量在感染后48 h后顯著降低(P0.01),NADH/NAD+比值顯著增高(P0.01)。說明IBDV感染DT40細胞后不但對線粒體造成了損傷,而且脂酰CoA的轉運和NADH呼吸鏈受到抑制,導致線粒體能量代謝障礙。本實驗從代謝角度為進一步探索IBDV感染對雞細胞的致病機理提供依據(jù)。2、IBDV感染對雞胚肝臟細胞線粒體能量代謝的影響為了進一步研究IBDV感染對宿主線粒體能量代謝的影響和從代謝方面揭示致病機制,本研究通過用IBDV毒株感染雞胚,通過傳代培養(yǎng),分離肝臟細胞線粒體,測定線粒體蛋白含量及線粒體呼吸鏈相關酶活性。結果表明,IBDV感染雞胚后,在傳代初期線粒體蛋白和ATP含量顯著降低(P0.01),肉堿脂酰轉移酶和NADH-Co Q還原酶活性顯著下降(P0.01);脂酰-CoA和ATP含量在感染后先顯著降低(P0.01);隨著傳代和雞胚適應,線粒體蛋白、ATP含量、肉堿脂酰轉移酶、NADH-Co Q還原酶活性以及脂酰-CoA逐漸升高,NADH/NAD+比值較傳代初期降低(P0.05)。比較而言,傳代初期丙酮酸水平顯著降低(P0.01),乳酸升高(P0.01),糖酵解途徑有加強趨勢,但細胞總ATP含量降低。實驗從機體層面證明了感染IBDV后,可以引起線粒體功能的缺陷,導致能量代謝的紊亂,影響雞胚的正常生長以至死亡。3、IBDV-VP4蛋白真核表達載體的構建和表達及其對DT40細胞線粒體能量代謝的影響為研究IBDV VP4蛋白與肉堿脂酰轉移酶結構擬態(tài)對脂酰-CoA轉運和NADH-CoQ還原酶及線粒體能量代謝的關系,本實驗通過擴增IBDV-VP4基因,構建真核表達載體pcDNA3.0-VP4并轉染DT40細胞,通過對VP4蛋白表達和線粒體進行能量代謝檢測,發(fā)現(xiàn)在轉染48 h后脂酰-CoA和ATP含量顯著升高(P0.01),肉堿脂酰轉移酶活性在轉染后72 h達到最高水平(P0.01);NADH/NAD+比值在48 h時顯著升高(P0.01),轉染中后期NADH-CoQ還原酶活性降低,ATP含量有所下降,表明VP4蛋白對線粒體呼吸鏈起始價段就產生了抑制作用;轉染早期丙酮酸顯著降低(P0.01),乳酸升高(P0.01),糖酵解加強。為從分子層次上研究IBDV感染細胞的致病機理與線粒體能量代謝的生物氧化的關系奠定了基礎。
[Abstract]:Chicken infectious bursal disease (Infectious bursal disease, IBD) is an acute one caused by infectious bursal disease virus (Infections bursal diseas virus, IBDV)..IBDV of highly contagious infectious disease is replicated quickly after invasion of the host, causing a large number of bad death and apoptosis in the B lymphocyte, causing serious damage to the immune function of the chicken. The process of infectious bursal disease is a complex process of multifaceted interaction between host and pathogen. The molecular mechanism of IBD is not very clear at present. This study is intended to infect DT40 and non immune chicken embryos by IBDV and detect the metabolic kinetics and energy levels of mitochondria from the metabolic layer. The relationship between mitochondrial biological oxidation and pathogenicity of IBDV infected cells was revealed. By expressing the effect of IBDV-VP4 protein on the transport of lipoyl CoA, the influence of VP4 protein molecular mimicry on the energy metabolism of mitochondrial respiratory chain was investigated at the molecular level.1. The effect of IBDV infection on the energy metabolism of B lymphocyte lines was to study the host of IBDV infection to the host. The effect of immune cell mitochondria metabolism, the IBDV isolates were infected with DT40 cells in this experiment. The samples were sampled at 24 h, 48 h, 72 h and 96 h respectively after the virus infection. The cell counts were counted and the mitochondria were separated. The mitochondrial protein content, mitochondrial membrane potential, carnitine lipoyltransferase and NADH-CoQ reductase activity, ATP level, and mitochondrial lipoyl -CoA were detected. With the content of NADH and NAD+ and the ratio of NADH/NAD+, the effect of IBDV infection on the biological oxidation of mitochondria in the host cells was preliminarily evaluated. The results showed that after IBDV infection of DT40 cells 48 h, the mitochondrial protein content decreased significantly (P0.01), the membrane potential decreased, and the activity of carnitine lipoyltransferase and NADH-CoQ reductase began to decrease (P0.05) after the infection of the 24 h (P0.05), and 48 h showed after the infection. Decrease (P0.01); the content of lipoyl -CoA and ATP decreased significantly (P0.01) after 48 h infection (P0.01), and the ratio of NADH/NAD+ increased significantly (P0.01). It indicated that IBDV infected DT40 cells not only caused damage to mitochondria, but also the transport of lipoyl CoA and NADH respiratory chain were inhibited, resulting in mitochondrial energy metabolism disorder. This experiment was further from the metabolic point of view. To explore the mechanism of IBDV infection on the pathogenesis of chicken cells, the effects of.2, IBDV infection on mitochondrial energy metabolism in chicken embryo liver cells were investigated in order to further study the effect of IBDV infection on the energy metabolism of the host mitochondria and to reveal the pathogenic mechanism from metabolic aspects. This study was carried out by infecting chicken embryos with IBDV virus and separating the liver through subculture and isolation of the liver. Mitochondria, mitochondrial protein content and mitochondrial respiratory chain related enzyme activity were measured. The results showed that after IBDV infection, the content of mitochondrial protein and ATP decreased significantly (P0.01), and the activity of carnitine lipoyltransferase and NADH-Co Q reductase decreased significantly (P0.01), and the content of lipoyl -CoA and ATP decreased significantly (P0.01) after infection. With the adaptation of generation and chicken embryo, mitochondrial protein, ATP content, carnitine lipoyltransferase, NADH-Co Q reductase activity and lipoyl -CoA gradually increased, NADH/NAD+ ratio decreased (P0.05) at the initial stage (P0.05). In comparison, the level of pyruvic acid decreased significantly (P0.01), lactic acid increased (P0.01), glycolysis pathway strengthened, but cell total AT The P content decreased. The experiment showed that after IBDV infection, the mitochondrial function was caused by the deficiency of mitochondrial function, the disturbance of the energy metabolism, the normal growth of the chicken embryo and the death of.3, the construction and expression of the eukaryotic expression vector of the IBDV-VP4 protein and the effect on the mitochondrial energy metabolism of DT40 cells and the study of the IBDV VP4 protein and carnitine fat. The relationship between the structure of acyltransferase and the structure of the acyl transferase on the transport of lipoyl -CoA and the energy metabolism of NADH-CoQ reductase and mitochondria. In this experiment, the IBDV-VP4 gene was amplified and the eukaryotic expression vector pcDNA3.0-VP4 was constructed and transfected to DT40 cells. The expression of VP4 protein and the energy metabolism of mitochondria were detected. The content of lipoyl -CoA and ATP after transfection of 48 h was found to be significant. The activity of carnitine lipoyl transferase reached the highest level (P0.01) at 72 h after transfection (P0.01), and the ratio of NADH/NAD+ increased significantly at 48 h (P0.01). The activity of NADH-CoQ reductase was decreased and the content of ATP decreased in the middle and late transfection. It indicated that the VP4 protein inhibited the initial segment of the mitochondrial respiratory chain, and the early pyruvic acid was significant in the transfection. Decrease (P0.01), increase of lactic acid (P0.01) and glycolysis strengthen. It lays a foundation for the study of the relationship between the pathogenesis of IBDV infected cells and the biological oxidation of mitochondrial energy metabolism at the molecular level.
【學位授予單位】：河南科技大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S858.31

【參考文獻】

相關期刊論文 前10條

1 李澤君;呼丹;熊克朝;吳純啟;丁日高;王茜莎;王全軍;;活性氧與線粒體損傷研究概述[J];中南藥學;2014年10期

2 李鳳杰;沈麗君;方合志;白益東;;線粒體呼吸鏈復合體Ⅰ[J];中國細胞生物學學報;2014年08期

3 張穎;劉鍇;劉艷春;;傳染性法氏囊病病毒研究進展[J];中國家禽;2013年24期

4 吳憶春;;雞傳染性法氏囊病病毒PCR檢測及VP4基因序列分析[J];動物醫(yī)學進展;2013年07期

5 吳媛媛;王宇祥;李輝;;雞肝臟內脂肪代謝相關因子的研究進展[J];畜牧與獸醫(yī);2013年01期

6 李小迪;覃楊;何蓉蓉;栗原博;;雞胚氧化應激模型研究進展[J];中國藥理學通報;2011年01期

7 羅俊;滕蔓;樊劍鳴;邢廣旭;李喬木;楊艷艷;張改平;;雞傳染性法氏囊病病毒在DT40細胞中的增殖規(guī)律[J];畜牧獸醫(yī)學報;2009年08期

8 劉樹森;;線粒體呼吸鏈與活性氧[J];生命科學;2008年04期

9 董志姚;李秀芬;劉立明;堵國成;陳堅;;過量表達NADH氧化酶加速光滑球擬酵母合成丙酮酸[J];微生物學報;2008年08期

10 李銀聚;吳庭才;張春杰;程相朝;余祖華;陳溥言;;IBDV不同野毒株雞胚傳代及各變異代次回歸雞后的致病性[J];中國獸醫(yī)學報;2008年05期

相關博士學位論文 前3條

1 王永志;傳染性法氏囊病毒感染的生物標記與細胞SAGE文庫的鑒定[D];浙江大學;2009年

2 鄭肖娟;傳染性法氏囊病病毒感染細胞的差異蛋白質組學研究[D];浙江大學;2007年

3 黃其春;甜菜堿對肥育豬脂肪代謝及其關鍵酶基因表達的影響與機理研究[D];浙江大學;2006年

相關碩士學位論文 前1條

1 熊怡;桿狀病毒誘導SL-1細胞凋亡過程中線粒體結構變化和凋亡相關蛋白的定位[D];華中師范大學;2010年

，

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