【摘要】：狂犬病是嗜神經(jīng)性狂犬病病毒(RV)感染哺乳動物中樞神經(jīng)系統(tǒng)而引起的急性、致死性傳染病,造成全球每年有50,000—59,000人死亡,但病例主要集中在亞洲和非洲。在狂犬病病毒感染的野生和家養(yǎng)宿主中,狗仍然被視為亞洲和非洲狂犬病的主要宿主。該病毒經(jīng)傷口感染后,沿周圍神經(jīng)逆軸漿侵入中樞,引起細胞凋亡、氧化應激,線粒體功能障礙,并通過劫持TLR3而抑制宿主的先天性免疫反應。應用狂犬病病毒固定株的體內(nèi)外神經(jīng)元感染實驗表明,該病毒引起樹突和軸突的退行性改變,而這些病理變化與神經(jīng)元細胞骨架密切相關。細胞骨架對細胞除起結構支撐作用外,微管和微絲動態(tài)變化還調(diào)節(jié)神經(jīng)細胞生長、物質運輸和細胞信號傳導等多種功能。然而,無論神經(jīng)元的兩種細胞骨架單獨或協(xié)同作用,均受構成這兩種骨架的骨架相關蛋白嚴格調(diào)控,特別是維持神經(jīng)元的正常結構和樹突棘形態(tài),在該過程中一些關鍵蛋白起主要作用。樹突棘是維持神經(jīng)正常傳導功能和興奮性突觸后膜的結構基礎,肌動蛋白為其主要骨架類型�？袢〔《窘柚W(wǎng)格蛋白實現(xiàn)內(nèi)吞并侵入細胞,其后形成的早期和晚期囊泡蛋白通過與肌動蛋白和微管的交互作用,完成侵襲過程,導致神經(jīng)元壞死和變性,因此出現(xiàn)神經(jīng)功能異常。鑒于狂犬病病毒和一些其他病毒經(jīng)肌動蛋白-微管骨架借助內(nèi)體途徑傳輸,因此,本研究旨在觀察狂犬病病毒感染后肌動蛋白和微管相關結合蛋白的動態(tài)變化及早、晚期囊泡上的GTP酶蛋白與狂犬病病毒的共定位等,分析狂犬病病毒在神經(jīng)元內(nèi)的傳輸機制,為狂犬病病毒感染導致的神經(jīng)功能異常提供實驗數(shù)據(jù)。本研究分為三部分。第一部分,感染神經(jīng)元細胞,然后用Rab5、EEA1、LAMP1、Rab7等囊泡表面特異性標記物抗體,免疫熒光檢測這些標志抗體全部或其中一些與RV是否存在共定位,并通過RNA干擾下調(diào)這些標志分子,研究其對于RV感染的影響。第二部分,在RV感染的神經(jīng)元上,應用實時定量PCR和Western blot印跡對微絲結合蛋白和微管相關蛋白,特別是微管正極端結合EB3和p140cap等進行了基因和蛋白表達水平檢測,并應用免疫熒光觀察了這些變化對樹突和軸突骨架的影響。在第三部分,應用免疫組織化學和組織病理學方法,對RV感染小鼠腦組織不同部位的抗原分布和病理變化進行了研究。同時還對肌動蛋白結合蛋白也進行了定量。這些研究結果可用于分析RV街毒株感染神經(jīng)元的胞體和周質變化。所有數(shù)據(jù)分別均采用SPSS和ANOVA,以確定對照組和實驗組間的統(tǒng)計學差異,相對定量分析以大于2倍作為顯著性范圍。結果表明,RV核蛋白與Rab5和Rab7在N2a細胞的細胞質中共定位,RNA干擾下調(diào)Rab5和Rab7表達時,在感染早期核蛋白和RV滴度均顯著降低。實時定量PCR檢測發(fā)現(xiàn),微管末端蛋白(EB3和p140cap)和肌動蛋白相關蛋白(pfn1,tesk1和LIMK1)均下調(diào),肌動蛋白相關蛋白(cfll 3,GSN,PPP3CA,SSH1,katnal)等上調(diào)。兩個不同RV毒株RV感染均顯著下調(diào)EB3和p140cap基因表達并減少其相應的蛋白含量,造成微管連續(xù)性破壞,呈散亂分布。組織病理學和免疫組化顯示,小鼠大腦不同區(qū)域均有病毒抗原分布,大腦可作為病理診斷或病料采集的最佳點。組織病理顯示,神經(jīng)元細胞輕度壞死和空泡化。這些結果說明狂犬病病毒通過改變細胞骨架相關蛋白而造成骨架結構被破壞,而從引起神經(jīng)元退化和神經(jīng)功能異常。本研究結果初步提示,狂犬病病毒感染使微管和肌動蛋白相關蛋白下調(diào),進而引起細胞骨架結構的完整性被破壞,造成神經(jīng)元變性和退行性改變,從而引起突觸功能障礙,表現(xiàn)出神經(jīng)功能異常。
[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.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S855.3
，

本文編號：2255601