本文關(guān)鍵詞：血紅素加氧酶1在牛病毒性腹瀉病毒感染MDBK細(xì)胞中的作用研究　出處：《西北農(nóng)林科技大學(xué)》2016年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 血紅素加氧酶1 牛病毒性腹瀉病毒 MDBK細(xì)胞 病原-細(xì)胞相互作用

【摘要】：牛病毒性腹瀉(Bovine viral diarrhea,BVD)是由牛病毒性腹瀉病毒(Bovine viral diarrhea virus,BVDV)感染引起的一種以腹瀉、急/慢性黏膜炎、繁殖障礙、持續(xù)性感染和免疫耐受為特征的傳染病。BVDV為單股正鏈RNA病毒,它與豬瘟病毒、羊邊界病病毒同屬于黃病毒科瘟病毒屬。該病毒具有廣泛的宿主嗜性,除了感染牛,還可感染豬、羊、鹿、駱駝及其它多種動(dòng)物并引起發(fā)病,使其在自然界中難以清除,給整個(gè)牲畜養(yǎng)殖業(yè)造成巨大的危害。BVDV感染懷孕母畜常引起免疫耐受,導(dǎo)致持續(xù)性感染(Persistent infection,PI)牛的產(chǎn)生。PI牛對(duì)疫苗免疫不產(chǎn)生相應(yīng)的抗體,病毒得以在體內(nèi)持續(xù)存在并不斷向外排毒感染其它牛群,這使得PI牛成為BVD感染中最主要的傳染源�，F(xiàn)有的防控策略很難將BVDV從牛群中根除,因此,我們亟需尋找一種新的抗病毒方案以有效防控BVDV的感染。近期研究發(fā)現(xiàn),血紅素加氧酶1(Heme oxygenase-1,HO-1)作為細(xì)胞中的一種重要的抗氧化應(yīng)激蛋白,不僅具有抗炎、抗氧化、抗凋亡等多種生物學(xué)功能,而且在病毒感染中也發(fā)揮著非常重要的調(diào)控作用。乙型肝炎病毒、丙型肝炎病毒、豬繁殖與呼吸綜合征病毒等病毒感染可以抑制HO-1的表達(dá),上調(diào)HO-1表達(dá)可有效抑制這些病毒的感染。但是豬瘟病毒感染卻可促進(jìn)HO-1的表達(dá),降低HO-1的表達(dá)反而抑制了該病毒的感染,這說(shuō)明不同病毒的感染,HO-1所發(fā)揮的作用并不相同。BVDV感染能否調(diào)控細(xì)胞中HO-1的表達(dá)?HO-1在BVDV感染細(xì)胞的過(guò)程中發(fā)揮什么作用?這一切都尚未清楚。因此本研究擬明確BVDV感染對(duì)宿主細(xì)胞HO-1表達(dá)的影響,進(jìn)一步闡明HO-1在BVDV感染細(xì)胞過(guò)程中的作用,主要研究結(jié)果如下:1.BVDV感染降低MDBK細(xì)胞中HO-1的表達(dá)水平我們首先檢測(cè)了BVDV感染后不同時(shí)間點(diǎn)MDBK細(xì)胞中BVDV、HO-1、HO-2、NQO1(NAD(P)H醌氧化還原酶1,與HO-1同屬于抗氧化反應(yīng)元件所調(diào)控)mRNA和蛋白的表達(dá)情況。結(jié)果表明,BVDV感染后MDBK細(xì)胞中HO-1及NQO1的表達(dá)水平顯著下降,且下降的趨勢(shì)與病毒感染時(shí)間呈正相關(guān),而HO-2的表達(dá)無(wú)顯著變化。這說(shuō)明BVDV感染可抑制MDBK細(xì)胞中HO-1表達(dá),其抑制作用初步證實(shí)與抗氧化通路相關(guān),而HO-2的表達(dá)不受影響。2.上調(diào)HO-1表達(dá)顯著抑制BVDV的感染為了研究HO-1在BVDV感染過(guò)程中的作用,我們首先應(yīng)用HO-1的誘導(dǎo)劑CoPP處理MBDK細(xì)胞,然后檢測(cè)CoPP誘導(dǎo)HO-1對(duì)BVDV復(fù)制的影響。結(jié)果表明,CoPP誘導(dǎo)顯著上調(diào)了細(xì)胞中HO-1的表達(dá)而HO-2表達(dá)水平?jīng)]有變化,并且細(xì)胞的活性不受影響。CoPP呈濃度梯度依賴(lài)性的降低BVDV mRNA和蛋白的表達(dá)水平及病毒滴度,而且不論病毒感染前或感染后用CoPP處理MDBK細(xì)胞,均可顯著降低BVDV的復(fù)制。該結(jié)果表明,CoPP誘導(dǎo)HO-1上調(diào)表達(dá)可有效抑制BVDV的感染。為證明CoPP對(duì)BVDV感染的抑制作用是由HO-1介導(dǎo)的,我們構(gòu)建了可表達(dá)HO-1的重組腺病毒,利用該病毒感染MDBK細(xì)胞,在細(xì)胞中特異性過(guò)表達(dá)HO-1,研究其對(duì)BVDV感染的影響。結(jié)果表明,感染重組腺病毒的細(xì)胞中HO-1的表達(dá)水平顯著升高,而B(niǎo)VDV的NS5B蛋白含量及細(xì)胞上清中病毒RNA的含量和病毒滴度均呈顯著降低。該結(jié)果證實(shí)了特異性上調(diào)HO-1的表達(dá)可有效抑制細(xì)胞中BVDV的感染。3.特異性降低HO-1基礎(chǔ)表達(dá)水平促進(jìn)BVDV的感染由于誘導(dǎo)或過(guò)表達(dá)HO-1均顯著降低了BVDV的感染,我們想進(jìn)一步了解降低細(xì)胞中HO-1的基礎(chǔ)表達(dá)水平能否影響B(tài)VDV的復(fù)制。因此我們將特異性干擾HO-1表達(dá)的siRNA轉(zhuǎn)入MDBK細(xì)胞以降低細(xì)胞中HO-1的基礎(chǔ)表達(dá)水平,然后檢測(cè)HO-1和BVDV mRNA和蛋白表達(dá)。結(jié)果表明,轉(zhuǎn)染siRNA的細(xì)胞HO-1的表達(dá)水平顯著降低,而細(xì)胞上清中病毒RNA的含量及病毒滴度卻顯著升高。說(shuō)明特異性降低HO-1的基礎(chǔ)表達(dá)水平可促進(jìn)BVDV的感染。4.CoPP通過(guò)HO-1介導(dǎo)抑制BVDV的復(fù)制為了證明CoPP處理對(duì)BVDV感染的抑制作用是由HO-1特異性介導(dǎo)的,轉(zhuǎn)染siRNA的MDBK細(xì)胞,接毒后用CoPP處理,然后檢測(cè)BVDV的病毒滴度和蛋白表達(dá)情況。結(jié)果表明,轉(zhuǎn)染siRNA可部分逆轉(zhuǎn)CoPP誘導(dǎo)對(duì)BVDV感染的抑制作用,該結(jié)果證明CoPP是通過(guò)HO-1的介導(dǎo)抑制BVDV的復(fù)制。5.HO-1抑制BVDV復(fù)制的作用與其代謝產(chǎn)物有關(guān)為進(jìn)一步了解HO-1抑制BVDV感染的原因,我們采用biliverdin、CORM-2及FeCl3(模擬HO-1的代謝產(chǎn)物biliverdin,CO和Fe2+)分別處理細(xì)胞,然后檢測(cè)BVDV的感染情況。結(jié)果表明,采用biliverdin和CORM-2處理的細(xì)胞,BVDV的感染均顯著降低,而采用FeCl3處理的細(xì)胞,BVDV感染無(wú)顯著變化,這初步證明了HO-1抑制BVDV感染的作用與其代謝產(chǎn)物biliverdin和CO有關(guān)。綜上所述,本研究證明了BVDV感染可以抑制MDBK細(xì)胞中HO-1的表達(dá),而上調(diào)HO-1表達(dá)可顯著抑制BVDV的感染,其抑制作用與HO-1的代謝產(chǎn)物biliverdin和CO相關(guān)。該研究為闡明BVDV的分子致病機(jī)制提供了有用的參考資料,并為抗BVDV感染的研究提供了新的思路及潛在的靶點(diǎn)。
[Abstract]:Bovine viral diarrhea (Bovine viral diarrhea, BVD) from bovine viral diarrhea virus (Bovine viral diarrhea virus, BVDV) an infection caused by diarrhea, acute / chronic mucositis, reproductive disorders, persistent infection and immune tolerance of infectious disease.BVDV positive single strand RNA virus, it classical swine fever virus, border disease virus belonging to the Flaviviridae pestivirus genus. The virus has a wide host tropism, apart from infected cows can also infect pigs, sheep, deer, camels and other animal and cause disease, making it difficult to remove in nature, for the livestock breeding industry great harm.BVDV infection of pregnant female often cause immune tolerance, cause persistent infection (Persistent infection, PI.PI) cattle cattle do not produce antibodies to the vaccine, the virus can persist in vivo and continue to infect other cattle from the pigs, the The PI become the main source of infectious bovine infection in BVD. The existing prevention and control strategy is very difficult to eradicate BVDV from the herd, therefore, we need to find a new antivirus solutions to effective prevention and control of BVDV infection. A recent study found that heme oxygenase 1 (Heme oxygenase-1 HO-1) is a kind of important oxidative stress proteins in the cell, not only has the anti-inflammatory, antioxidant, anti apoptosis of various biological functions, but also in the viral infection also plays a very important role in the regulation of hepatitis B virus, hepatitis C virus, porcine reproductive and respiratory syndrome virus can inhibit the expression of HO-1 infection, can effectively inhibit the upregulation of the expression of HO-1 the virus infection. But the swine fever virus infection can promote the expression of HO-1, decreased the expression of HO-1 may suppress the virus infection, indicating that different virus infection, HO-1 plays the role of Is not the same as the.BVDV infection in HO-1 cells can express HO-1? What is the role of BVDV in the infected cells? All this is not clear. Therefore, this study intends to clear effect of BVDV infection on the expression of host cell HO-1, to further elucidate the role of HO-1 in BVDV infected cells in the process, the main results are as follows: 1.BVDV the infection decreased the expression of HO-1 in MDBK cells, we first detected the infection of BVDV BVDV at different time points after MDBK cells in HO-1, HO-2, NQO1 (NAD (P) H quinone oxidoreductase 1, and HO-1 belong to the antioxidant response element regulated) expression of mRNA and protein. The results showed that the expression level of BVDV infection after MDBK and HO-1 in NQO1 cells decreased significantly, and decreased with the infection time was positively related to the expression of HO-2 showed no significant changes. This shows that BVDV infection can inhibit the expression of HO-1 in MDBK cells and its inhibition Preliminary confirmed with antioxidant pathway, whereas the expression of HO-2 was not affected by the expression of.2. up-regulated HO-1 significantly inhibited BVDV infection in order to study the HO-1 during BVDV infection, the induction of CoPP MBDK cells we applied HO-1, then detect the effect of HO-1 on CoPP induced BVDV replication. The results showed that CoPP induced significant upregulation of the expression of HO-1 in the cells and the expression level of HO-2 did not change, and the activity of the cells was not affected by.CoPP concentration gradient dependent and virus titer of BVDV decreased the expression of mRNA and protein, and no virus infection before or after infection with CoPP MDBK cells, BVDV could significantly decrease the replication of the results. Show that the expression of BVDV can effectively inhibit the infection of HO-1 induced upregulation of CoPP. In order to prove the inhibitory effect of CoPP on BVDV infection is mediated by HO-1, we constructed the recombinant adenovirus expressing HO-1 , the virus infected MDBK cells, the cell specific expression of HO-1, to study its effect on BVDV infection. The results showed that the HO-1 expression level of recombinant adenovirus infected cells significantly increased, while the content of RNA and the virus titer of virus NS5B protein and BVDV in the cell supernatant were significantly reduced. The results confirm the specificity of the expression regulation of HO-1 infected.3. specific cells can effectively inhibit the decrease of BVDV in HO-1 based BVDV expression level to promote infection due to induction or overexpression of HO-1 decreased significantly in BVDV infection, we want to know more about reducing the expression level of HO-1 in cells can influence the replication of BVDV. So we the specificity of siRNA expression by HO-1 interference into MDBK cells to reduce the expression level of HO-1 in cells, and the detection of HO-1 and BVDV mRNA and protein expression. The results showed that the transfection of siRNA fine The expression level of HO-1 cells decreased significantly, while the content of virus and virus titer in the supernatant of RNA cells was significantly increased. Lower specificity HO-1 based expression can promote the infection of.4.CoPP BVDV through HO-1 mediated inhibition of BVDV replication in order to prove that CoPP treatment of BVDV infection is inhibited by HO-1 mediated by specific the siRNA transfected MDBK cells after inoculation with CoPP, then the virus titer of BVDV and protein expression. The results showed that the transfection of siRNA could reverse CoPP induced inhibition of BVDV infection, the results show that CoPP inhibition of BVDV replication and its metabolites related to further understand the inhibition of HO-1 BVDV infection by HO-1 mediated inhibition of BVDV.5.HO-1 replication, we use biliverdin, CORM-2 and FeCl3 (analog HO-1 metabolites of biliverdin, CO and Fe2+) were treated cells, then detected The infection of BVDV. The results showed that the biliverdin and CORM-2 treated cells, BVDV infection were significantly decreased, while the use of FeCl3 treated cells, no significant changes of BVDV infection, which demonstrated that HO-1 inhibited the role of BVDV infection and its metabolites biliverdin and CO. In conclusion, this study demonstrated that HO-1 can inhibit the expression of MDBK cells in BVDV infection, and overexpression of HO-1 can inhibit BVDV infection, and the inhibitory effect of HO-1 metabolites biliverdin and CO related. This study provides a useful reference for the molecular pathogenesis of BVDV is clarified, and provides a new idea and a potential target for the study of anti BVDV infection.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：S855.3
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本文編號(hào)：1362306