本文選題：傳染性法氏囊病毒 + 線粒體��； 參考：《河南科技大學(xué)》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?gòu)?fù)制致使B淋巴細(xì)胞大量壞死和凋亡,導(dǎo)致雞的免疫功能受到嚴(yán)重?fù)p傷,造成被感染家禽嚴(yán)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(fù)雜過程,目前IBD致病的分子機(jī)制并不十分清楚。本研究擬以IBDV感染DT40細(xì)胞和非免疫雞胚,通過對(duì)線粒體代謝動(dòng)力學(xué)和能量水平的檢測(cè),從代謝層面上揭示IBDV感染細(xì)胞的線粒體生物氧化與致病性的關(guān)系;通過表達(dá)IBDV-VP4蛋白對(duì)脂酰CoA轉(zhuǎn)運(yùn)的影響,從分子層面探討VP4蛋白分子擬態(tài)對(duì)線粒體呼吸鏈能量代謝的影響。1、IBDV感染對(duì)B淋巴細(xì)胞線粒體能量代謝的影響為研究IBDV感染對(duì)宿主免疫細(xì)胞線粒體代謝的影響,本實(shí)驗(yàn)以IBDV分離株感染DT40細(xì)胞,分別于病毒感染后24 h、48 h、72 h和96 h取樣,細(xì)胞計(jì)數(shù)并分離細(xì)胞線粒體,通過檢測(cè)線粒體蛋白含量,線粒體膜電位,肉堿脂酰轉(zhuǎn)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(píng)估IBDV感染對(duì)宿主細(xì)胞線粒體生物氧化的影響。結(jié)果表明,IBDV感染DT40細(xì)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(zhuǎn)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(xì)胞后不但對(duì)線粒體造成了損傷,而且脂酰CoA的轉(zhuǎn)運(yùn)和NADH呼吸鏈?zhǔn)艿揭种?導(dǎo)致線粒體能量代謝障礙。本實(shí)驗(yàn)從代謝角度為進(jìn)一步探索IBDV感染對(duì)雞細(xì)胞的致病機(jī)理提供依據(jù)。2、IBDV感染對(duì)雞胚肝臟細(xì)胞線粒體能量代謝的影響為了進(jìn)一步研究IBDV感染對(duì)宿主線粒體能量代謝的影響和從代謝方面揭示致病機(jī)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(xì)胞線粒體,測(cè)定線粒體蛋白含量及線粒體呼吸鏈相關(guān)酶活性。結(jié)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(zhuǎn)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(yīng),線粒體蛋白、ATP含量、肉堿脂酰轉(zhuǎn)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(qiáng)趨勢(shì),但細(xì)胞總ATP含量降低。實(shí)驗(yàn)從機(jī)體層面證明了感染IBDV后,可以引起線粒體功能的缺陷,導(dǎo)致能量代謝的紊亂,影響雞胚的正常生長(zhǎng)以至死亡。3、IBDV-VP4蛋白真核表達(dá)載體的構(gòu)建和表達(dá)及其對(duì)DT40細(xì)胞線粒體能量代謝的影響為研究IBDV VP4蛋白與肉堿脂酰轉(zhuǎn)移酶結(jié)構(gòu)擬態(tài)對(duì)脂酰-CoA轉(zhuǎn)運(yùn)和NADH-CoQ還原酶及線粒體能量代謝的關(guān)系,本實(shí)驗(yàn)通過擴(kuò)增IBDV-VP4基因,構(gòu)建真核表達(dá)載體pcDNA3.0-VP4并轉(zhuǎn)染DT40細(xì)胞,通過對(duì)VP4蛋白表達(dá)和線粒體進(jìn)行能量代謝檢測(cè),發(fā)現(xiàn)在轉(zhuǎn)染48 h后脂酰-CoA和ATP含量顯著升高(P0.01),肉堿脂酰轉(zhuǎn)移酶活性在轉(zhuǎn)染后72 h達(dá)到最高水平(P0.01);NADH/NAD+比值在48 h時(shí)顯著升高(P0.01),轉(zhuǎn)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(duì)線粒體呼吸鏈起始價(jià)段就產(chǎn)生了抑制作用;轉(zhuǎn)染早期丙酮酸顯著降低(P0.01),乳酸升高(P0.01),糖酵解加強(qiáng)。為從分子層次上研究IBDV感染細(xì)胞的致病機(jī)理與線粒體能量代謝的生物氧化的關(guān)系奠定了基礎(chǔ)。
[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(xué)位授予單位】：河南科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S858.31

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本文編號(hào)：1933124