狂犬病病毒感染导致微管和微丝相关蛋白表达水平改变
发布时间：2018-10-07 20:40
【摘要】：狂犬病是嗜神经性狂犬病病毒(RV)感染哺乳动物中枢神经系统而引起的急性、致死性传染病,造成全球每年有50,000—59,000人死亡,但病例主要集中在亚洲和非洲。在狂犬病病毒感染的野生和家养宿主中,狗仍然被视为亚洲和非洲狂犬病的主要宿主。该病毒经伤口感染后,沿周围神经逆轴浆侵入中枢,引起细胞凋亡、氧化应激,线粒体功能障碍,并通过劫持TLR3而抑制宿主的先天性免疫反应。应用狂犬病病毒固定株的体内外神经元感染实验表明,该病毒引起树突和轴突的退行性改变,而这些病理变化与神经元细胞骨架密切相关。细胞骨架对细胞除起结构支撑作用外,微管和微丝动态变化还调节神经细胞生长、物质运输和细胞信号传导等多种功能。然而,无论神经元的两种细胞骨架单独或协同作用,均受构成这两种骨架的骨架相关蛋白严格调控,特别是维持神经元的正常结构和树突棘形态,在该过程中一些关键蛋白起主要作用。树突棘是维持神经正常传导功能和兴奋性突触后膜的结构基础,肌动蛋白为其主要骨架类型。狂犬病病毒借助网格蛋白实现内吞并侵入细胞,其后形成的早期和晚期囊泡蛋白通过与肌动蛋白和微管的交互作用,完成侵袭过程,导致神经元坏死和变性,因此出现神经功能异常。鉴于狂犬病病毒和一些其他病毒经肌动蛋白-微管骨架借助内体途径传输,因此,本研究旨在观察狂犬病病毒感染后肌动蛋白和微管相关结合蛋白的动态变化及早、晚期囊泡上的GTP酶蛋白与狂犬病病毒的共定位等,分析狂犬病病毒在神经元内的传输机制,为狂犬病病毒感染导致的神经功能异常提供实验数据。本研究分为三部分。第一部分,感染神经元细胞,然后用Rab5、EEA1、LAMP1、Rab7等囊泡表面特异性标记物抗体,免疫荧光检测这些标志抗体全部或其中一些与RV是否存在共定位,并通过RNA干扰下调这些标志分子,研究其对于RV感染的影响。第二部分,在RV感染的神经元上,应用实时定量PCR和Western blot印迹对微丝结合蛋白和微管相关蛋白,特别是微管正极端结合EB3和p140cap等进行了基因和蛋白表达水平检测,并应用免疫荧光观察了这些变化对树突和轴突骨架的影响。在第三部分,应用免疫组织化学和组织病理学方法,对RV感染小鼠脑组织不同部位的抗原分布和病理变化进行了研究。同时还对肌动蛋白结合蛋白也进行了定量。这些研究结果可用于分析RV街毒株感染神经元的胞体和周质变化。所有数据分别均采用SPSS和ANOVA,以确定对照组和实验组间的统计学差异,相对定量分析以大于2倍作为显著性范围。结果表明,RV核蛋白与Rab5和Rab7在N2a细胞的细胞质中共定位,RNA干扰下调Rab5和Rab7表达时,在感染早期核蛋白和RV滴度均显著降低。实时定量PCR检测发现,微管末端蛋白(EB3和p140cap)和肌动蛋白相关蛋白(pfn1,tesk1和LIMK1)均下调,肌动蛋白相关蛋白(cfll 3,GSN,PPP3CA,SSH1,katnal)等上调。两个不同RV毒株RV感染均显著下调EB3和p140cap基因表达并减少其相应的蛋白含量,造成微管连续性破坏,呈散乱分布。组织病理学和免疫组化显示,小鼠大脑不同区域均有病毒抗原分布,大脑可作为病理诊断或病料采集的最佳点。组织病理显示,神经元细胞轻度坏死和空泡化。这些结果说明狂犬病病毒通过改变细胞骨架相关蛋白而造成骨架结构被破坏,而从引起神经元退化和神经功能异常。本研究结果初步提示,狂犬病病毒感染使微管和肌动蛋白相关蛋白下调,进而引起细胞骨架结构的完整性被破坏,造成神经元变性和退行性改变,从而引起突触功能障碍,表现出神经功能异常。
[Abstract]:Rabies are acute and fatal infectious diseases caused by the infection of the central nervous system of a mammalian nervous rabies virus (RV), resulting in the death of 50,000 or 59,000 people a year, but the case is mainly concentrated in Asia and Africa. Dogs are still considered a major host of rabies in Asia and Africa in the wild and wild hosts of rabies virus infections. After wound infection, the virus invaded the center along the reverse axis of peripheral nerve, causing cell apoptosis, oxidative stress, mitochondrial dysfunction, and inhibited the host's innate immune response by hijacking TLR3. The experimental results show that the virus causes degeneration of the dendritic and axons, and these pathological changes are closely related to the neuronal apoptosis. The dynamic changes of microtubules and microfilaments also regulate nerve cell growth, substance transport and cell signal transduction in addition to structural support. However, regardless of the individual or synergistic effects of the neurons, the skeleton-related proteins of the two frameworks are regulated strictly, especially the normal structure and dendritic spines of the neurons, and some of the key proteins play a major role in the process. The dendritic spines are the structural bases for maintaining the normal conduction function of the nerves and the excitable synaptic membranes, and actin is the main skeleton type. Rabies virus (Rabies Virus) is involved in the invasion and invasion of cells by means of fusion protein, and the early and late vesicle proteins are formed by interaction with actin and microtubules, which leads to neuronal necrosis and degeneration, resulting in neurological abnormalities. in view of the fact that the rabies virus and some other viruses are transported via the inner body pathway via the actin-microtubule framework, the present study is intended to observe the early dynamic changes of actin and microtubule-associated binding proteins after rabies virus infection, The transmission mechanism of rabies virus in neurons is analyzed by the co-localization of the GTP enzyme protein and the rabies virus on the late vesicle, and experimental data are provided for the neurological function abnormality caused by rabies virus infection. This study is divided into three parts. the first part, the infected neuron cell, and then uses the vesicle surface specific marker antibody of Rab5, EEA1, LAMP1, Rab7 to detect whether all or some of the marker antibodies are co-located with the RV and down-regulate the marker molecules through RNA interference, Study its effect on RV infection. In the second part, real-time quantitative PCR and Western blot were applied to detect the gene and protein expression level of microfilament binding protein and microtubule-related protein, especially tubulin terminal binding EB3 and p140so on the neurons infected by RV. Immunofluorescence was applied to observe the effect of these changes on the dendritic and axons. In the third part, immunohistochemical and histopathological methods were applied to study the antigenic distribution and pathological changes of different parts of the brain tissue of RV infected mice. The actin-binding protein was also quantified. These results can be used to analyze the cellular and periplasmic changes of the RV strain infected neurons. SPSS and ANOVA were used for all data to determine the statistical difference between the control group and the experimental group, and the relative quantitative analysis was greater than 2 times as the significance range. The results showed that RV nucleoprotein was co-located with Rab5 and Rab7 in the cytoplasm of N2a cells, RNA interference reduced Rab5 and Rab7 expression, and the early protein and RV drop were significantly decreased during the infection. Real-time quantitative PCR detection revealed that tubulin-related proteins (EB3 and p1405B) and actin-related proteins (CDn1, teesk1 and LIMK1) were downregulated, and actin-related proteins (cfll 3, GSN, PP3M CA, SSH1, karnal) were upregulated. RV infection of two different RV strains significantly downregulated the expression of EB3 and p140ARF genes and reduced their corresponding protein content, which resulted in the disruption of microtubules and scattered distribution. Histopathology and immunohistochemistry show that there are virus antigens in different regions of the brain, and the brain can be used as the best spot for pathological diagnosis or disease collection. Histopathology revealed mild necrosis and vacuolization of neuronal cells. These results suggest that the rabies virus results in the destruction of the skeletal structure by altering the tau-related protein and from causing neuronal degeneration and neurological dysfunction. The results of this study suggest that rabies virus infection causes the down-regulation of tubulin and actin-related proteins, which leads to the destruction of the structural integrity, resulting in neuronal degeneration and degenerative changes, resulting in synaptic dysfunction, showing neurological abnormalities.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S855.3
，
本文编号：2255601
[Abstract]:Rabies are acute and fatal infectious diseases caused by the infection of the central nervous system of a mammalian nervous rabies virus (RV), resulting in the death of 50,000 or 59,000 people a year, but the case is mainly concentrated in Asia and Africa. Dogs are still considered a major host of rabies in Asia and Africa in the wild and wild hosts of rabies virus infections. After wound infection, the virus invaded the center along the reverse axis of peripheral nerve, causing cell apoptosis, oxidative stress, mitochondrial dysfunction, and inhibited the host's innate immune response by hijacking TLR3. The experimental results show that the virus causes degeneration of the dendritic and axons, and these pathological changes are closely related to the neuronal apoptosis. The dynamic changes of microtubules and microfilaments also regulate nerve cell growth, substance transport and cell signal transduction in addition to structural support. However, regardless of the individual or synergistic effects of the neurons, the skeleton-related proteins of the two frameworks are regulated strictly, especially the normal structure and dendritic spines of the neurons, and some of the key proteins play a major role in the process. The dendritic spines are the structural bases for maintaining the normal conduction function of the nerves and the excitable synaptic membranes, and actin is the main skeleton type. Rabies virus (Rabies Virus) is involved in the invasion and invasion of cells by means of fusion protein, and the early and late vesicle proteins are formed by interaction with actin and microtubules, which leads to neuronal necrosis and degeneration, resulting in neurological abnormalities. in view of the fact that the rabies virus and some other viruses are transported via the inner body pathway via the actin-microtubule framework, the present study is intended to observe the early dynamic changes of actin and microtubule-associated binding proteins after rabies virus infection, The transmission mechanism of rabies virus in neurons is analyzed by the co-localization of the GTP enzyme protein and the rabies virus on the late vesicle, and experimental data are provided for the neurological function abnormality caused by rabies virus infection. This study is divided into three parts. the first part, the infected neuron cell, and then uses the vesicle surface specific marker antibody of Rab5, EEA1, LAMP1, Rab7 to detect whether all or some of the marker antibodies are co-located with the RV and down-regulate the marker molecules through RNA interference, Study its effect on RV infection. In the second part, real-time quantitative PCR and Western blot were applied to detect the gene and protein expression level of microfilament binding protein and microtubule-related protein, especially tubulin terminal binding EB3 and p140so on the neurons infected by RV. Immunofluorescence was applied to observe the effect of these changes on the dendritic and axons. In the third part, immunohistochemical and histopathological methods were applied to study the antigenic distribution and pathological changes of different parts of the brain tissue of RV infected mice. The actin-binding protein was also quantified. These results can be used to analyze the cellular and periplasmic changes of the RV strain infected neurons. SPSS and ANOVA were used for all data to determine the statistical difference between the control group and the experimental group, and the relative quantitative analysis was greater than 2 times as the significance range. The results showed that RV nucleoprotein was co-located with Rab5 and Rab7 in the cytoplasm of N2a cells, RNA interference reduced Rab5 and Rab7 expression, and the early protein and RV drop were significantly decreased during the infection. Real-time quantitative PCR detection revealed that tubulin-related proteins (EB3 and p1405B) and actin-related proteins (CDn1, teesk1 and LIMK1) were downregulated, and actin-related proteins (cfll 3, GSN, PP3M CA, SSH1, karnal) were upregulated. RV infection of two different RV strains significantly downregulated the expression of EB3 and p140ARF genes and reduced their corresponding protein content, which resulted in the disruption of microtubules and scattered distribution. Histopathology and immunohistochemistry show that there are virus antigens in different regions of the brain, and the brain can be used as the best spot for pathological diagnosis or disease collection. Histopathology revealed mild necrosis and vacuolization of neuronal cells. These results suggest that the rabies virus results in the destruction of the skeletal structure by altering the tau-related protein and from causing neuronal degeneration and neurological dysfunction. The results of this study suggest that rabies virus infection causes the down-regulation of tubulin and actin-related proteins, which leads to the destruction of the structural integrity, resulting in neuronal degeneration and degenerative changes, resulting in synaptic dysfunction, showing neurological abnormalities.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S855.3
，
本文编号：2255601
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