自噬对不同生物型牛病毒性腹泻病毒感染宿主细胞的作用研究

发布时间：2018-06-04 10:07

本文选题：细胞自噬 + 牛病毒性腹泻病毒　；参考：《吉林农业大学》2017年硕士论文

【摘要】：细胞自噬是一种真核生物的进化保守的过程,自噬的主要功能是降解内源性蛋白质等生物大分子以实现氨基酸和单糖的再循环。当细胞内缺乏营养时自噬起到的内环境稳态的作用是十分重要。自噬在生物学和医学等其他各个领域都是飞速发展的探究热点,国际上第一篇文章于2004年在Science上发表,国内在2007年开始对自噬的研究。国内研究主要集中于人类医学,动物医学方面研究鲜有。牛病毒性腹泻病毒(Bovine Viral Diarrhea Virus,BVDV),又名牛病毒性腹泻-黏膜病病毒(Bovine Viral Diarrhea-Mucosal Disease,BVD-MD),属于黄病毒科瘟病毒属的成员。牛病毒性腹泻病毒根据不同的5‘UTR区域可以将BVDV分为基因Ⅰ型和Ⅱ型,同时也可以根据病毒在细胞内致细胞病变效应分为致细胞病变型(CPE)和非致细胞病变型(nCPE)两个型。本研究从蛋白、分子及细胞形态学等方面对不同生物型BVDV病毒与宿主细胞自噬相互关系进行了研究,并应用药物作用对比,初步阐述了不同生物型BVDV病毒作用宿主细胞MDBK后,病毒复制与自噬相互的关系。通过透射电镜和转染荧光质粒GFP-LC3的方法分别检测和观察感染病毒的细胞内的自噬泡和自噬体。在透射电镜下,感染病毒的细胞内自噬泡数量明显多于正常细胞,感染CPE型病毒的细胞内的自噬泡多于感染nCPE型病毒的细胞内自噬泡的数量。通过转染荧光质粒的试验发现感染病毒的细胞内自噬体数量明显多于正常细胞内的自噬体,感染CPE型病毒的细胞内的自噬体多于感染nCPE型的细胞内自噬体的数量。随着感染时间的延长,感染病毒的细胞内自噬泡和自噬体的数量都会有所增加。在自噬研究的过程当中,单单通过评价自噬体以及自噬泡的数量或LC3和P62的表达均不能代表整个自噬系统活化,必须检测动态自噬流的变化才能更全面客观地评价自噬活性。因此,在自噬的检测过程中必须是对自噬流的检测才能更好地说明自噬的活性,得到的结果才能是可靠的指标。利用Western blot的方法检测LC3和P62的自噬流,将自噬相关药物自噬促进剂雷帕霉素和自噬的抑制剂3-MA作用于感染BVDV的细胞上,感染CPE型和nCPE型病毒的细胞内LC3-Ⅱ型的量均发生了明显的增加,感染CPE型病毒的细胞内LC3-Ⅱ型多于感染nCPE型的细胞内的量。并且感染CPE型的细胞内P62的量减少的量多于感染nCPE型的细胞内的量。BVDV CPE型相对于nCPE型促进自噬的效果更加明显。药物作用下感染病毒的细胞内LC3-Ⅱ的量的变化更加明显,蛋白P62的量也明显的减少。说明CPE型促进了自噬,而nCPE型则抑制了自噬。使用实时荧光定量PCR检测病毒复制的量。得到自噬在nCPE型复制的初期是抑制了该病毒的复制;而对于CPE型病毒,自噬则促进了其复制。自噬是机体的天然免疫反应,研究表明,细胞自噬具有高度的保守性,透射电镜结果显示,自噬体可直接捕获侵入机体的病原,并将之清除。本研究通过LC3-I/II和P62的检测,结果表明,nCPE型BVDV病原体虽不能逃避细胞自噬的识别,但可通过阻断自噬体降解和自噬流的产生,使病毒粒子富集在自噬体内,利用细胞自噬有利于其在细胞内复制,为自身的复制提供便利条件。而CPE型毒株对细胞自噬则起到活化和促进作用。使用自噬促进剂雷帕酶素和自噬抑制剂3-MA,与病毒对照进一步分析不同生物型病毒感染宿主细胞后自噬与病毒复制的关系,在不同时间点收取样品,qPCR进行病毒含量测定。自噬抑制剂3-MA作用宿主细胞后,接种病毒NM株,24h时,病毒复制量无明显差别,随着时间的推移,病毒对照组复制水平显著高于药物组,试验结果说明自噬功能受到抑制的同时,病毒复制受到抑制,宿主细胞自噬功能与NM株复制呈正相关。本研究对自噬与BVDV不同生物型病毒感染宿主细胞的相互作用关系及对病毒的复制影响进行了初步分析,为进一步深入研究BVDV免疫机制和持续性感染机制提供了理论支撑。
[Abstract]:Autophagy is an evolutionary conservative process of a eukaryotic organism. The main function of autophagy is to degrade biological macromolecules such as endogenous proteins to realize the recirculation of amino acids and monosaccharides. The role of autophagy in the homeostasis of autophagy in the absence of nutrients is very important. Autophagy is in many other fields, such as biology and medicine. It is the hot spot of rapid development. The first international article was published on Science in 2004 and the domestic research on autophagy began in 2007. Domestic research is mainly focused on human medicine. There are few studies on animal medicine. Bovine Viral Diarrhea Virus (BVDV), also known as bovine viral diarrhea mucous membrane virus (Bo). Vine Viral Diarrhea-Mucosal Disease, BVD-MD), belonging to the genus oryvirus. Bovine viral diarrhea virus can be divided into genotype I and type II according to different 5 'UTR regions, and can be divided into cytopathic type (CPE) and non cytopathic type (nCPE) two according to the cytopathic effect of the virus in the cell. The relationship between the autophagy of different biotype BVDV viruses and host cells was studied from the aspects of protein, molecular and cell morphology. The relationship between the host cell MDBK of different biotype BVDV viruses and the relationship between virus replication and autophagy was preliminarily expounded. Transmission electron microscopy and transfection of fluoro were carried out. In the transmission electron microscope, the number of autophagic vacuoles in the cells infected by the virus is obviously more than that of the normal cells. The number of autophagic vesicles in the cells infected with CPE virus is more than that of the intracellular autophagic vesicles infected with the nCPE virus. By transfecting the fluorescent light quality, the number of autophagic vacuoles in the infected cells is more than that in the cells infected with the virus. It was found that the number of autophagic bodies in the cells infected by the virus was significantly more than the autophagic body in the normal cells. The autophagic bodies in the cells infected with the CPE virus were more than the number of autophagic bodies in the cells infected with nCPE. The number of autophagic and autophagic in the cells infected with the virus increased with the time of infection. In the course of the study, the autophagic body, the number of autophagic vesicles, or the expression of LC3 and P62 can not represent the entire autophagic system activation. It is necessary to detect the dynamic autophagic flow to evaluate the autophagy activity more comprehensively and objectively. Therefore, the autophagy detection process must be better explained by the detection of autophagic flow. The results of autophagy can be a reliable indicator. The autophagy flow of LC3 and P62 was detected by Western blot, and the autophagy related drug autophagy promoter, rapamycin and autophagy inhibitor 3-MA, was acted on the cells infected with BVDV, and the amount of LC3- II in the cells infected with the CPE and nCPE virus increased significantly. The number of intracellular LC3- II infected with CPE virus is more than that in the cells infected with nCPE type. And the amount of P62 in the cells infected with CPE is more than that in the cells infected with the nCPE type. The.BVDV CPE type is more effective than the nCPE type to promote autophagy. The changes in the amount of LC3- II in the cells of the infected cells are more evident. The amount of protein P62 also decreased significantly. It indicated that the CPE type promoted autophagy, while the nCPE type inhibited autophagy. The amount of virus replication was detected by real-time fluorescence quantitative PCR. Autophagy was inhibited in the early stage of nCPE replication; for the CPE virus, autophagy promoted its replication. Autophagy was the natural immune reaction of the body. The study shows that autophagy is highly conserved, and transmission electron microscope results show that autophagic can directly capture the pathogens that invade the body and remove it. The results of the study by LC3-I/II and P62 showed that the nCPE type BVDV pathogen could not escape the recognition of autophagy, but could block the degradation of autophagic and autophagy by blocking the autophagy. It is produced by enriching the virus particles in autophagy, using autophagy to facilitate its replication in cells and providing convenience for its own replication. The CPE strain plays an active and promoting role in the autophagy of cells. The autophagy promoter Rapa and the autophagy inhibitor 3-MA are used to further analyze different biologic viruses from the virus. The relationship between autophagy and virus replication after infection of the host cells, samples were collected at different time points and qPCR was measured. After the autophagy inhibitor 3-MA acted host cell, the virus replication amount was not significantly different when the virus NM strain was inoculated and 24h, the replication level of the virus was significantly higher than that of the drug group as time went on, and the test results showed that the virus replication level was significantly higher than that of the drug group. The autophagy was inhibited and the replication of the virus was inhibited. The autophagy of the host cell was positively related to the replication of the NM strain. The interaction of autophagy with the host cells infected by BVDV different biotype viruses and the effect on the replication of the virus were preliminarily analyzed in this study to further study the BVDV immune mechanism and continue to be sexy. The dyeing mechanism provides theoretical support.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.653

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