【摘要】：狂犬病是由狂犬病病毒（RABV）引起的一種以神經(jīng)系統(tǒng)感染為特征的烈性、致死性人獸共患傳染病。發(fā)病動物有明顯的神經(jīng)癥狀，如：恐水、恐聲、驚厥等。在RABV感染的成年鼠腦組織切片中能夠看到軸突、樹突呈串珠樣改變和腫脹，這些變化與神經(jīng)元細胞骨架結構的改變密切相關。微管作為細胞內最重要的骨架類型，其結構的穩(wěn)定性直接決定了細胞的正常功能。為研究神經(jīng)元細胞微管和RABV致病性的關系，本研究在觀察到RABV感染引起神經(jīng)元微管結構改變的基礎上，通過檢測影響微管穩(wěn)定性的相關蛋白在轉錄，翻譯及細胞內分布的變化，研究RABV感染與神經(jīng)元微管相關蛋白變化的關系，這些研究可為對RABV感染致神經(jīng)功能異退化機制的研究提供理論依據(jù)，并為進一步研究RABV發(fā)病機制奠定基礎。 本研究首先通過對乳鼠腦內接種RABV固定毒株進行病毒擴增，并測定病毒滴度。隨后對能夠影響細胞骨架穩(wěn)定性的相關基因進行篩選，包括EB3、p140cap、Rbl2、Tppp、Vasp、Fascin1、Tesk2、GIT2及Ena，并從中挑選變化明顯的轉錄異常基因進行下一步研究。之后通過實時定量PCR、Western blot及免疫熒光對基因轉錄水平、蛋白水平、細胞內分布進行檢測，然后通過Westernblot檢測其下游蛋白的活性。最后，以該基因在CVS感染神經(jīng)元內的異常表達為基礎，研究RABV街毒株MRV對該基因轉錄、蛋白及細胞內分布水平是否有相似影響，以進一步探究狂犬病病毒街毒株致神經(jīng)元功能異常的機制。 結果顯示，對挑選的9個微管相關基因的實時定量PCR檢測發(fā)現(xiàn)，EB3、p140cap、Rbl2、Tppp、Vasp、Fascin1、Tesk2及Ena等8個出現(xiàn)轉錄異常，從中挑選2個異常的相關基因EB3、p140cap及p140cap下游蛋白Rac1進行研究。首先對CVS感染不同時間神經(jīng)元內EB3轉錄和翻譯水平的動態(tài)變化進行實時定量PCR及Western Blot檢測。結果顯示，，CVS感染1h后，與對照組相比，EB3mRNA量和蛋白量都有明顯的降低。且隨著時間延長，mRNA量和蛋白量進一步降低，與對照組差異更加明顯。免疫熒光實驗結果顯示，CVS感染48h，神經(jīng)元胞體內有少量病毒特異性光斑，EB3較連續(xù)地分布于突起；感染96h，在胞體和突起病毒光斑增多，EB3呈斑狀雜亂地分布于突起及胞體膜內側，失去在正常神經(jīng)元上的點狀分布狀態(tài)。對p140cap進行動態(tài)檢測發(fā)現(xiàn)，CVS感染1h之后，在轉錄及翻譯水平上也有明顯的下降。對p140cap下游蛋白Rac1活性檢測發(fā)現(xiàn)CVS感染能夠明顯抑制Rac1的活性。對MRV感染的神經(jīng)元EB3、p140cap轉錄、表達水平及分布檢測，證實EB3的轉錄及翻譯水平下調。在感染96h后，EB3呈散沙狀均勻分布于突起上。同時還發(fā)現(xiàn)，MRV感染能抑制p140cap轉錄，但對其蛋白表達下調作用不明顯。 通過本研究，可以得出以下結論：1、CVS感染引起EB3（↓）、p140cap（↓）、p130（↓）、Tppp（↓）、GIT2（↓）、Fascin1（↓）、Ena（↓）及Tesk2（↑）等基因轉錄異常，抑制EB3及p140cap的表達，改變EB3細胞內定位，從而影響神經(jīng)元MTs的穩(wěn)定性。CVS感染對Rac1活性的抑制，可能通過EB3—p140cap—Rac1信號途徑而影響微絲的重排。2、RABV街毒株MRV能夠抑制EB3表達并改變其細胞內定位，對p140cap表達影響不明顯。
[Abstract]:Rabies is caused by rabies virus (RABV) caused by a nervous system infection characterized by a strong, fatal zoonosis. The animals have obvious neurological symptoms, such as water threatening, phobia, convulsion, etc. in the brain tissue of RABV infected adult rats, the axons can be seen, the dendrites are beaded and swollen, and these changes It is closely related to the changes in the cytoskeleton structure of neurons. As the most important skeleton type in the cell, the stability of the microtubule directly determines the normal function of the cells. In order to study the relationship between the microtubule and the pathogenicity of RABV, this study is based on the observation of the changes in the microtubule structure of the neuron caused by RABV infection through examination. The changes in the transcription, translation and intracellular distribution of related proteins affecting the stability of microtubule were measured, and the relationship between RABV infection and the changes of neuron microtubule related proteins could be studied. These studies can provide a theoretical basis for the study of the mechanism of RABV infection induced neurofunction degradation, and lay a foundation for the further study of the pathogenesis of RABV.
In this study, we first amplified the virus by inoculating RABV in the brain of the rat and tested the virus titer. Subsequently, the genes that could affect the stability of cytoskeleton were screened, including EB3, p140cap, Rbl2, Tppp, Vasp, Fascin1, Tesk2, GIT2 and Ena, and the next step was to be selected for the next step in the selection of abnormal transcriptional genes. Then the gene transcriptional level, protein level, and intracellular distribution were detected by real-time quantitative PCR, Western blot and immunofluorescence, and then the activity of the downstream protein was detected by Westernblot. Finally, based on the abnormal expression of the gene in the CVS infected neurons, the RABV Street strain MRV was studied in the transcription, protein and cell of the gene. Whether the distribution level has a similar effect to further explore the mechanism of neuronal dysfunction induced by rabies virus street virus.
The results showed that EB3, p140cap, Rbl2, Tppp, Vasp, Fascin1, Tesk2 and Ena were detected by real time quantitative PCR detection of the selected 9 microtubule related genes, and selected 2 abnormal genes, EB3, p140cap and downstream downstream proteins. Real-time quantitative PCR and Western Blot detection were performed on the dynamic changes of the level. The results showed that after CVS infection was 1H, the amount of EB3mRNA and protein decreased significantly compared with the control group. And with the time prolonging, the amount of mRNA and protein decreased further, and the difference was more obvious with the control group. The results of immunofluorescence test showed that CVS infected 48h, neuron cell There are a few virus specific spots in the body, EB3 is more continuous in the protuberance, the infection 96h, the increase of the light spots in the cell and the protuberance virus, the EB3 distribution in the protuberance and the inner membrane of the cell membrane, and the distribution of the dot like distribution on the normal neurons. The dynamic test of p140cap shows that after the CVS infection of 1H, the transcriptional and translation levels are at the level. The detection of the downstream protein Rac1 activity of p140cap found that CVS infection could significantly inhibit the activity of Rac1. EB3, p140cap transcription, expression level and distribution detection of MRV infected neurons confirmed that the transcription and translation level of EB3 was downregulated. After infection 96h, EB3 was distributed evenly on the protuberance. Meanwhile, MRV sense was found. Staining can inhibit p140cap transcription, but its down-regulation effect on protein expression is not obvious.
Through this study, we can draw the following conclusions: 1, CVS infection causes EB3, p130, Tppp, GIT2, Ena, Tesk2, and Tesk2, to inhibit the expression of EB3 and p140cap, and to alter the localization of the EB3 cells, which may affect the inhibition of the activity of the infected neurons. Through the EB3 - p140cap - Rac1 signal pathway, the rearrangement of microfilament.2, RABV Street strain MRV can inhibit the expression of EB3 and change its intracellular location, and the effect on p140cap expression is not obvious.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R512.99

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