本文選題：細胞自噬 + 牛病毒性腹瀉病毒　； 參考：《吉林農業(yè)大學》2017年碩士論文

【摘要】：細胞自噬是一種真核生物的進化保守的過程,自噬的主要功能是降解內源性蛋白質等生物大分子以實現(xiàn)氨基酸和單糖的再循環(huán)。當細胞內缺乏營養(yǎng)時自噬起到的內環(huán)境穩(wěn)態(tài)的作用是十分重要。自噬在生物學和醫(yī)學等其他各個領域都是飛速發(fā)展的探究熱點,國際上第一篇文章于2004年在Science上發(fā)表,國內在2007年開始對自噬的研究。國內研究主要集中于人類醫(yī)學,動物醫(yī)學方面研究鮮有。牛病毒性腹瀉病毒(Bovine Viral Diarrhea Virus,BVDV),又名牛病毒性腹瀉-黏膜病病毒(Bovine Viral Diarrhea-Mucosal Disease,BVD-MD),屬于黃病毒科瘟病毒屬的成員。牛病毒性腹瀉病毒根據(jù)不同的5‘UTR區(qū)域可以將BVDV分為基因Ⅰ型和Ⅱ型,同時也可以根據(jù)病毒在細胞內致細胞病變效應分為致細胞病變型(CPE)和非致細胞病變型(nCPE)兩個型。本研究從蛋白、分子及細胞形態(tài)學等方面對不同生物型BVDV病毒與宿主細胞自噬相互關系進行了研究,并應用藥物作用對比,初步闡述了不同生物型BVDV病毒作用宿主細胞MDBK后,病毒復制與自噬相互的關系。通過透射電鏡和轉染熒光質粒GFP-LC3的方法分別檢測和觀察感染病毒的細胞內的自噬泡和自噬體。在透射電鏡下,感染病毒的細胞內自噬泡數(shù)量明顯多于正常細胞,感染CPE型病毒的細胞內的自噬泡多于感染nCPE型病毒的細胞內自噬泡的數(shù)量。通過轉染熒光質粒的試驗發(fā)現(xiàn)感染病毒的細胞內自噬體數(shù)量明顯多于正常細胞內的自噬體,感染CPE型病毒的細胞內的自噬體多于感染nCPE型的細胞內自噬體的數(shù)量。隨著感染時間的延長,感染病毒的細胞內自噬泡和自噬體的數(shù)量都會有所增加。在自噬研究的過程當中,單單通過評價自噬體以及自噬泡的數(shù)量或LC3和P62的表達均不能代表整個自噬系統(tǒng)活化,必須檢測動態(tài)自噬流的變化才能更全面客觀地評價自噬活性。因此,在自噬的檢測過程中必須是對自噬流的檢測才能更好地說明自噬的活性,得到的結果才能是可靠的指標。利用Western blot的方法檢測LC3和P62的自噬流,將自噬相關藥物自噬促進劑雷帕霉素和自噬的抑制劑3-MA作用于感染BVDV的細胞上,感染CPE型和nCPE型病毒的細胞內LC3-Ⅱ型的量均發(fā)生了明顯的增加,感染CPE型病毒的細胞內LC3-Ⅱ型多于感染nCPE型的細胞內的量。并且感染CPE型的細胞內P62的量減少的量多于感染nCPE型的細胞內的量。BVDV CPE型相對于nCPE型促進自噬的效果更加明顯。藥物作用下感染病毒的細胞內LC3-Ⅱ的量的變化更加明顯,蛋白P62的量也明顯的減少。說明CPE型促進了自噬,而nCPE型則抑制了自噬。使用實時熒光定量PCR檢測病毒復制的量。得到自噬在nCPE型復制的初期是抑制了該病毒的復制;而對于CPE型病毒,自噬則促進了其復制。自噬是機體的天然免疫反應,研究表明,細胞自噬具有高度的保守性,透射電鏡結果顯示,自噬體可直接捕獲侵入機體的病原,并將之清除。本研究通過LC3-I/II和P62的檢測,結果表明,nCPE型BVDV病原體雖不能逃避細胞自噬的識別,但可通過阻斷自噬體降解和自噬流的產生,使病毒粒子富集在自噬體內,利用細胞自噬有利于其在細胞內復制,為自身的復制提供便利條件。而CPE型毒株對細胞自噬則起到活化和促進作用。使用自噬促進劑雷帕酶素和自噬抑制劑3-MA,與病毒對照進一步分析不同生物型病毒感染宿主細胞后自噬與病毒復制的關系,在不同時間點收取樣品,qPCR進行病毒含量測定。自噬抑制劑3-MA作用宿主細胞后,接種病毒NM株,24h時,病毒復制量無明顯差別,隨著時間的推移,病毒對照組復制水平顯著高于藥物組,試驗結果說明自噬功能受到抑制的同時,病毒復制受到抑制,宿主細胞自噬功能與NM株復制呈正相關。本研究對自噬與BVDV不同生物型病毒感染宿主細胞的相互作用關系及對病毒的復制影響進行了初步分析,為進一步深入研究BVDV免疫機制和持續(xù)性感染機制提供了理論支撐。
[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.
【學位授予單位】：吉林農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.653

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