狂犬病病毒感染抑制神经元内微管相关蛋白EB3及p140cap表达的研究

发布时间：2018-08-04 18:30

【摘要】：狂犬病是由狂犬病病毒（RABV）引起的一种以神经系统感染为特征的烈性、致死性人兽共患传染病。发病动物有明显的神经症状，如：恐水、恐声、惊厥等。在RABV感染的成年鼠脑组织切片中能够看到轴突、树突呈串珠样改变和肿胀，这些变化与神经元细胞骨架结构的改变密切相关。微管作为细胞内最重要的骨架类型，其结构的稳定性直接决定了细胞的正常功能。为研究神经元细胞微管和RABV致病性的关系，本研究在观察到RABV感染引起神经元微管结构改变的基础上，通过检测影响微管稳定性的相关蛋白在转录，翻译及细胞内分布的变化，研究RABV感染与神经元微管相关蛋白变化的关系，这些研究可为对RABV感染致神经功能异退化机制的研究提供理论依据，并为进一步研究RABV发病机制奠定基础。本研究首先通过对乳鼠脑内接种RABV固定毒株进行病毒扩增，并测定病毒滴度。随后对能够影响细胞骨架稳定性的相关基因进行筛选，包括EB3、p140cap、Rbl2、Tppp、Vasp、Fascin1、Tesk2、GIT2及Ena，并从中挑选变化明显的转录异常基因进行下一步研究。之后通过实时定量PCR、Western blot及免疫荧光对基因转录水平、蛋白水平、细胞内分布进行检测，然后通过Westernblot检测其下游蛋白的活性。最后，以该基因在CVS感染神经元内的异常表达为基础，研究RABV街毒株MRV对该基因转录、蛋白及细胞内分布水平是否有相似影响，以进一步探究狂犬病病毒街毒株致神经元功能异常的机制。结果显示，对挑选的9个微管相关基因的实时定量PCR检测发现，EB3、p140cap、Rbl2、Tppp、Vasp、Fascin1、Tesk2及Ena等8个出现转录异常，从中挑选2个异常的相关基因EB3、p140cap及p140cap下游蛋白Rac1进行研究。首先对CVS感染不同时间神经元内EB3转录和翻译水平的动态变化进行实时定量PCR及Western Blot检测。结果显示，，CVS感染1h后，与对照组相比，EB3mRNA量和蛋白量都有明显的降低。且随着时间延长，mRNA量和蛋白量进一步降低，与对照组差异更加明显。免疫荧光实验结果显示，CVS感染48h，神经元胞体内有少量病毒特异性光斑，EB3较连续地分布于突起；感染96h，在胞体和突起病毒光斑增多，EB3呈斑状杂乱地分布于突起及胞体膜内侧，失去在正常神经元上的点状分布状态。对p140cap进行动态检测发现，CVS感染1h之后，在转录及翻译水平上也有明显的下降。对p140cap下游蛋白Rac1活性检测发现CVS感染能够明显抑制Rac1的活性。对MRV感染的神经元EB3、p140cap转录、表达水平及分布检测，证实EB3的转录及翻译水平下调。在感染96h后，EB3呈散沙状均匀分布于突起上。同时还发现，MRV感染能抑制p140cap转录，但对其蛋白表达下调作用不明显。通过本研究，可以得出以下结论：1、CVS感染引起EB3（↓）、p140cap（↓）、p130（↓）、Tppp（↓）、GIT2（↓）、Fascin1（↓）、Ena（↓）及Tesk2（↑）等基因转录异常，抑制EB3及p140cap的表达，改变EB3细胞内定位，从而影响神经元MTs的稳定性。CVS感染对Rac1活性的抑制，可能通过EB3—p140cap—Rac1信号途径而影响微丝的重排。2、RABV街毒株MRV能够抑制EB3表达并改变其细胞内定位，对p140cap表达影响不明显。
[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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R512.99

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