IBDV感染对宿主细胞线粒体能量代谢及致病性的关系研究

发布时间：2018-05-25 10:37

本文选题：传染性法氏囊病毒 + 线粒体　；参考：《河南科技大学》2017年硕士论文

【摘要】：鸡传染性法氏囊病(Infectious bursal disease,IBD)是由传染性法氏囊病病毒(Infections bursal diseas virus,IBDV)引起的鸡的一种急性、高度接触性传染病。IBDV在入侵宿主后迅速复制致使B淋巴细胞大量坏死和凋亡,导致鸡的免疫功能受到严重损伤,造成被感染家禽严重的免疫抑制。传染性法氏囊病的发生过程,是宿主与病原多层面作用的复杂过程,目前IBD致病的分子机制并不十分清楚。本研究拟以IBDV感染DT40细胞和非免疫鸡胚,通过对线粒体代谢动力学和能量水平的检测,从代谢层面上揭示IBDV感染细胞的线粒体生物氧化与致病性的关系;通过表达IBDV-VP4蛋白对脂酰CoA转运的影响,从分子层面探讨VP4蛋白分子拟态对线粒体呼吸链能量代谢的影响。1、IBDV感染对B淋巴细胞线粒体能量代谢的影响为研究IBDV感染对宿主免疫细胞线粒体代谢的影响,本实验以IBDV分离株感染DT40细胞,分别于病毒感染后24 h、48 h、72 h和96 h取样,细胞计数并分离细胞线粒体,通过检测线粒体蛋白含量,线粒体膜电位,肉碱脂酰转移酶和NADH-CoQ还原酶活性,ATP水平,线粒体脂酰-CoA和NADH、NAD+的含量及NADH/NAD+比值,初步评估IBDV感染对宿主细胞线粒体生物氧化的影响。结果表明,IBDV感染DT40细胞48 h后,线粒体蛋白含量显著降低(P0.01),膜电位下降,肉碱脂酰转移酶和NADH-CoQ还原酶活性在感染后24 h开始下降(P0.05),48 h后显著降低(P0.01);脂酰-CoA和ATP含量在感染后48 h后显著降低(P0.01),NADH/NAD+比值显著增高(P0.01)。说明IBDV感染DT40细胞后不但对线粒体造成了损伤,而且脂酰CoA的转运和NADH呼吸链受到抑制,导致线粒体能量代谢障碍。本实验从代谢角度为进一步探索IBDV感染对鸡细胞的致病机理提供依据。2、IBDV感染对鸡胚肝脏细胞线粒体能量代谢的影响为了进一步研究IBDV感染对宿主线粒体能量代谢的影响和从代谢方面揭示致病机制,本研究通过用IBDV毒株感染鸡胚,通过传代培养,分离肝脏细胞线粒体,测定线粒体蛋白含量及线粒体呼吸链相关酶活性。结果表明,IBDV感染鸡胚后,在传代初期线粒体蛋白和ATP含量显著降低(P0.01),肉碱脂酰转移酶和NADH-Co Q还原酶活性显著下降(P0.01);脂酰-CoA和ATP含量在感染后先显著降低(P0.01);随着传代和鸡胚适应,线粒体蛋白、ATP含量、肉碱脂酰转移酶、NADH-Co Q还原酶活性以及脂酰-CoA逐渐升高,NADH/NAD+比值较传代初期降低(P0.05)。比较而言,传代初期丙酮酸水平显著降低(P0.01),乳酸升高(P0.01),糖酵解途径有加强趋势,但细胞总ATP含量降低。实验从机体层面证明了感染IBDV后,可以引起线粒体功能的缺陷,导致能量代谢的紊乱,影响鸡胚的正常生长以至死亡。3、IBDV-VP4蛋白真核表达载体的构建和表达及其对DT40细胞线粒体能量代谢的影响为研究IBDV VP4蛋白与肉碱脂酰转移酶结构拟态对脂酰-CoA转运和NADH-CoQ还原酶及线粒体能量代谢的关系,本实验通过扩增IBDV-VP4基因,构建真核表达载体pcDNA3.0-VP4并转染DT40细胞,通过对VP4蛋白表达和线粒体进行能量代谢检测,发现在转染48 h后脂酰-CoA和ATP含量显著升高(P0.01),肉碱脂酰转移酶活性在转染后72 h达到最高水平(P0.01);NADH/NAD+比值在48 h时显著升高(P0.01),转染中后期NADH-CoQ还原酶活性降低,ATP含量有所下降,表明VP4蛋白对线粒体呼吸链起始价段就产生了抑制作用;转染早期丙酮酸显著降低(P0.01),乳酸升高(P0.01),糖酵解加强。为从分子层次上研究IBDV感染细胞的致病机理与线粒体能量代谢的生物氧化的关系奠定了基础。
[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.
【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.31

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