發(fā)布時(shí)間：2018-06-04 10:07

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

【摘要】：細(xì)胞自噬是一種真核生物的進(jìn)化保守的過程,自噬的主要功能是降解內(nèi)源性蛋白質(zhì)等生物大分子以實(shí)現(xiàn)氨基酸和單糖的再循環(huán)。當(dāng)細(xì)胞內(nèi)缺乏營養(yǎng)時(shí)自噬起到的內(nèi)環(huán)境穩(wěn)態(tài)的作用是十分重要。自噬在生物學(xué)和醫(yī)學(xué)等其他各個(gè)領(lǐng)域都是飛速發(fā)展的探究熱點(diǎn),國際上第一篇文章于2004年在Science上發(fā)表,國內(nèi)在2007年開始對(duì)自噬的研究。國內(nèi)研究主要集中于人類醫(yī)學(xué),動(dòng)物醫(yī)學(xué)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(shí)也可以根據(jù)病毒在細(xì)胞內(nèi)致細(xì)胞病變效應(yīng)分為致細(xì)胞病變型(CPE)和非致細(xì)胞病變型(nCPE)兩個(gè)型。本研究從蛋白、分子及細(xì)胞形態(tài)學(xué)等方面對(duì)不同生物型BVDV病毒與宿主細(xì)胞自噬相互關(guān)系進(jìn)行了研究,并應(yīng)用藥物作用對(duì)比,初步闡述了不同生物型BVDV病毒作用宿主細(xì)胞MDBK后,病毒復(fù)制與自噬相互的關(guān)系。通過透射電鏡和轉(zhuǎn)染熒光質(zhì)粒GFP-LC3的方法分別檢測(cè)和觀察感染病毒的細(xì)胞內(nèi)的自噬泡和自噬體。在透射電鏡下,感染病毒的細(xì)胞內(nèi)自噬泡數(shù)量明顯多于正常細(xì)胞,感染CPE型病毒的細(xì)胞內(nèi)的自噬泡多于感染nCPE型病毒的細(xì)胞內(nèi)自噬泡的數(shù)量。通過轉(zhuǎn)染熒光質(zhì)粒的試驗(yàn)發(fā)現(xiàn)感染病毒的細(xì)胞內(nèi)自噬體數(shù)量明顯多于正常細(xì)胞內(nèi)的自噬體,感染CPE型病毒的細(xì)胞內(nèi)的自噬體多于感染nCPE型的細(xì)胞內(nèi)自噬體的數(shù)量。隨著感染時(shí)間的延長,感染病毒的細(xì)胞內(nèi)自噬泡和自噬體的數(shù)量都會(huì)有所增加。在自噬研究的過程當(dāng)中,單單通過評(píng)價(jià)自噬體以及自噬泡的數(shù)量或LC3和P62的表達(dá)均不能代表整個(gè)自噬系統(tǒng)活化,必須檢測(cè)動(dòng)態(tài)自噬流的變化才能更全面客觀地評(píng)價(jià)自噬活性。因此,在自噬的檢測(cè)過程中必須是對(duì)自噬流的檢測(cè)才能更好地說明自噬的活性,得到的結(jié)果才能是可靠的指標(biāo)。利用Western blot的方法檢測(cè)LC3和P62的自噬流,將自噬相關(guān)藥物自噬促進(jìn)劑雷帕霉素和自噬的抑制劑3-MA作用于感染BVDV的細(xì)胞上,感染CPE型和nCPE型病毒的細(xì)胞內(nèi)LC3-Ⅱ型的量均發(fā)生了明顯的增加,感染CPE型病毒的細(xì)胞內(nèi)LC3-Ⅱ型多于感染nCPE型的細(xì)胞內(nèi)的量。并且感染CPE型的細(xì)胞內(nèi)P62的量減少的量多于感染nCPE型的細(xì)胞內(nèi)的量。BVDV CPE型相對(duì)于nCPE型促進(jìn)自噬的效果更加明顯。藥物作用下感染病毒的細(xì)胞內(nèi)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(jìn)了自噬,而nCPE型則抑制了自噬。使用實(shí)時(shí)熒光定量PCR檢測(cè)病毒復(fù)制的量。得到自噬在nCPE型復(fù)制的初期是抑制了該病毒的復(fù)制;而對(duì)于CPE型病毒,自噬則促進(jìn)了其復(fù)制。自噬是機(jī)體的天然免疫反應(yīng),研究表明,細(xì)胞自噬具有高度的保守性,透射電鏡結(jié)果顯示,自噬體可直接捕獲侵入機(jī)體的病原,并將之清除。本研究通過LC3-I/II和P62的檢測(cè),結(jié)果表明,nCPE型BVDV病原體雖不能逃避細(xì)胞自噬的識(shí)別,但可通過阻斷自噬體降解和自噬流的產(chǎn)生,使病毒粒子富集在自噬體內(nèi),利用細(xì)胞自噬有利于其在細(xì)胞內(nèi)復(fù)制,為自身的復(fù)制提供便利條件。而CPE型毒株對(duì)細(xì)胞自噬則起到活化和促進(jìn)作用。使用自噬促進(jìn)劑雷帕酶素和自噬抑制劑3-MA,與病毒對(duì)照進(jìn)一步分析不同生物型病毒感染宿主細(xì)胞后自噬與病毒復(fù)制的關(guān)系,在不同時(shí)間點(diǎn)收取樣品,qPCR進(jìn)行病毒含量測(cè)定。自噬抑制劑3-MA作用宿主細(xì)胞后,接種病毒NM株,24h時(shí),病毒復(fù)制量無明顯差別,隨著時(shí)間的推移,病毒對(duì)照組復(fù)制水平顯著高于藥物組,試驗(yàn)結(jié)果說明自噬功能受到抑制的同時(shí),病毒復(fù)制受到抑制,宿主細(xì)胞自噬功能與NM株復(fù)制呈正相關(guān)。本研究對(duì)自噬與BVDV不同生物型病毒感染宿主細(xì)胞的相互作用關(guān)系及對(duì)病毒的復(fù)制影響進(jìn)行了初步分析,為進(jìn)一步深入研究BVDV免疫機(jī)制和持續(xù)性感染機(jī)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(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.653

【參考文獻(xiàn)】

相關(guān)期刊論文 前9條

1 宮曉煒;鄭福英;陳啟偉;劉永生;李兆才;周繼章;才學(xué)鵬;殷宏;;細(xì)胞自噬對(duì)牛病毒性腹瀉病毒復(fù)制的影響[J];畜牧獸醫(yī)學(xué)報(bào);2015年07期

2 朱斐;王徵;;細(xì)胞自噬與病毒復(fù)制[J];中國細(xì)胞生物學(xué)學(xué)報(bào);2014年05期

3 陶冶;任曉峰;;細(xì)胞自噬與病毒感染[J];病毒學(xué)報(bào);2013年01期

4 王海杰;譚玉珍;;細(xì)胞自噬研究技術(shù)進(jìn)展及其應(yīng)用[J];中國細(xì)胞生物學(xué)學(xué)報(bào);2011年07期

5 趙美;臧偉進(jìn);;巨自噬在心臟疾病調(diào)節(jié)中的雙向作用[J];生理科學(xué)進(jìn)展;2011年02期

6 王寵;張萍;朱衛(wèi)國;;細(xì)胞自噬與腫瘤發(fā)生的關(guān)系[J];中國生物化學(xué)與分子生物學(xué)報(bào);2010年11期

7 朱娜;譚文杰;;細(xì)胞自噬對(duì)人冠狀病毒復(fù)制與致病性的影響[J];病毒學(xué)報(bào);2010年06期

8 何韜;譚玉珍;王海杰;;自噬在巨噬細(xì)胞清除凋亡淋巴細(xì)胞中的作用[J];解剖學(xué)報(bào);2008年04期

9 王海杰;譚玉珍;;細(xì)胞自噬的形態(tài)學(xué)特征和功能意義[J];解剖學(xué)報(bào);2009年05期

相關(guān)博士學(xué)位論文 前1條

1 康愷;豬瘟病毒復(fù)制與細(xì)胞自噬關(guān)系的研究[D];西北農(nóng)林科技大學(xué);2015年

相關(guān)碩士學(xué)位論文 前1條

1 王淅鳳;自噬相關(guān)蛋白Beclin-1和p62在非小細(xì)胞肺癌中的表達(dá)及其臨床意義[D];蘇州大學(xué);2016年

，

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