【摘要】：豬流行性腹瀉(porcine epidemic diarrhea, PED)是由豬流行性腹瀉病毒(porcine epidemic diarrhea virus, PEDV)引起的一種以水樣腹瀉、嘔吐、脫水為主要特征的豬腸道傳染性疾病,給世界養(yǎng)豬業(yè)造成了巨大的經(jīng)濟損失。盡管對PEDV致病機制進行了一定的研究,取得了一系列有重要意義的成果,但在病毒的侵染機制及與宿主細胞之間的相互作用機制等方面,研究的較少,是近年來被廣泛研究的熱點。本研究主要針對HMGB1在PEDV感染中作用機制進行了深入探討,系統(tǒng)分析了 PEDV感染與宿主蛋白HMGB1相互關系的機制。主要研究內(nèi)容包括:1.甘草素對PEDV的抗病毒效果甘草素是一個來源于傳統(tǒng)中藥甘草的天然產(chǎn)物。通過western blot、熒光定量PCR和噬斑形成試驗證明了甘草素具有抑制PEDV感染Vero細胞的效果。此外,探索了甘草素抑制PEDV感染的作用過程,發(fā)現(xiàn)甘草素對PEDV的入胞和復制有影響,而對細胞、病毒粒子本身、病毒的組裝和釋放沒有影響。2. HMGB1在甘草素的抗病毒過程中起著重要的作用HMGB1是一個細胞中含量非常豐富的核蛋白,能作為一個重要的炎性介質(zhì)釋放到胞外空間調(diào)節(jié)宿主的炎癥反應。我們發(fā)現(xiàn)甘草素能降低由PEDV感染引起的促炎性因子(IL-1β,IL-6,IL-8,TNF-α)的升高,且Poly(I:C)引起促炎性因子的升高且促進了PEDV的感染。甘草素是HMGB1的競爭性抑制劑。通過HMGB1抗體中和試驗和siRNA干擾HMGB1表達,發(fā)現(xiàn)HMGB1在感染條件下對促炎性因子的升高起著重要的作用。HMGB1可與TLR4和RAGE等受體相結(jié)合,因此利用TLR4的抑制劑和RAGE的siRNA處理細胞,發(fā)現(xiàn)TLR4抑制劑和siRAGE能降低促炎性因子的升高和病毒的感染,且過表達HMGB1的突變體(PCA-C45S、PCA-C106S、PCA-C45106S)可降低促炎性因子的升高和PEDV的感染。此外,我們還證明了 HMGB1與TLR4和RAGE結(jié)合后可激活P38MAPK來誘導促炎性因子的升高進而有利于PEDV的感染�？傊�,甘草素與胞外HMGB1結(jié)合后,降低了其與TLR4和RAGE受體的結(jié)合,從而降低了P38MAPK的激活,使促炎性因子降低來抑制PEDV的增殖。3.硫酸類肝素是豬流行性腹瀉病毒感染Vero細胞的吸附因子許多病毒利用硫酸類肝素作為它們的吸附因子。在該研究中,證明了 PEDV利用硫酸類肝素吸附Vero細胞。Western blot分析、熒光定量PCR和噬斑形成試驗揭示被肝素預處理的病毒,共感染細胞的能力受到抑制;而用肝素預處理細胞,對PEDV的感染沒有抑制效應。其次,我們發(fā)現(xiàn)肝素酶Ⅰ處理細胞去除硫酸類肝素后,細胞對PEDV的易感性下降。另外,我們證明抑制硫酸類肝素生物合成的氯酸鈉處理細胞,能夠有效抑制PEDV的感染。我們還檢測了不同程度硫酸化的兩個肝素類似物對PEDV感染的影響,數(shù)據(jù)表明N-去硫酸化肝素(不是N-乙�；疧-去硫酸化肝素)預處理病毒能夠降低病毒感染細胞的能力。此外,肝素可抑制PEDV的復制和HMGB1 mRNA水平�？傊�,研究表明硫酸類肝素是PEDV感染Vero細胞的吸附因子。4. PEDV N蛋白通過與C/EBP-β相互作用來正調(diào)HMGB1的轉(zhuǎn)錄及乙�；歪尫臜MGB1是一個重要的促炎性介質(zhì)并且促進多種炎性疾病的發(fā)生。發(fā)現(xiàn)了 PEDV感染導致HMGB1的乙�；歪尫�,PEDV的感染和PEDV衣殼蛋白通過SIRT1、組蛋白乙酰轉(zhuǎn)移酶和NF-κB調(diào)節(jié)HMGB1的乙�；�。過表達PEDVN蛋白能促進HMGB1的乙酰化和釋放。CHIP試驗表明PEDVN能與HMGB1啟動子區(qū)DNA相互作用。雙熒光素酶報告基因試驗證明了 PEDVN蛋白介導的HMGB1的轉(zhuǎn)錄與轉(zhuǎn)錄因子C/EBP結(jié)合基序有關。用Co-IP試驗進一步表明病毒的衣殼蛋白與C/EBP-β相互作用來正調(diào)HMGB1基因的轉(zhuǎn)錄�？傊�,PEDV感染能引起HMGB1的乙�；歪尫�,且主要是PEDV N蛋白起作用。我們的結(jié)果對PEDV的發(fā)病提供了新的視角并且為發(fā)展以HMGB1為靶點的治療方法和藥物提供了理論基礎。5.豬流行性腹瀉病毒通過DNA損傷引起HMGB1的釋放由DNA病毒激活的DNA損傷反應被廣泛的研究。然而,RNA病毒引起DNA損傷反應的研究很少。因此通過western blot和間接免疫熒光揭示了 PEDV快速激活H2AX的磷酸化,且HY增加了 PEDVN蛋白的表達。此外,通過western blot、qRT-PCR和噬斑形成試驗證明DNA-PK抑制劑(NU7441)和ATM抑制劑(KU-55933 )抑制了 HMGB1的釋放和PEDV的感染,且DNA-PK的抑制劑效果更好。但是ATR的抑制劑(VE-821)對PEDV感染沒有影響。此外,PEDV引起PARP1的激活,PARP1的抑制劑3-AB抑制了 HMGB1的釋放及PEDV的感染。DNA-PK抑制劑(NU7441 )和ATM抑制劑(KU-55933)抑制了 PARP1的活性,表明PEDV主要通過誘導DNA-PK依賴的DNA損傷和部分ATM依賴的DNA損傷激活PARP1和PEDV的增殖。通過流式細胞術證明了 PEDV引起ROS的升高,且抑制DNA損傷和PARP1的激活能降低ROS�？傊�,PEDV通過DNA損傷/PARP1/ROS引起HMGB1的釋放從而有利于自身的復制。
[Abstract]:Porcine epidemic diarrhea (PED), which is caused by the porcine epidemic diarrhea virus (PEDV), is an infectious disease of the intestinal tract, which is characterized by watery diarrhea, vomiting and dehydration, and has caused great economic losses to the pig industry in the world. Although some research has been carried out on the pathogenesis of the PEDV, a series of important results have been obtained. However, the research on the mechanism of the infection of the virus and the mechanism of the interaction with the host cell is a hotspot in recent years. The mechanism of the relationship between the PEDV infection and the host protein HMGB1 was analyzed. The main research contents include:1. The antiviral effect of the glycyrrhizin on the PEDV is a natural product derived from the traditional Chinese medicine licorice. The results of western blot, fluorescence quantitative PCR and plaque formation demonstrated that the glycyrrhizin had the effect of inhibiting the infection of Vero cells with PEDV. In addition, the effect of glycyrrhizin on the inhibition of PEDV infection was explored, and it was found that the glycyrrhizin had an effect on the cell and replication of the PEDV, and no effect on the cell, viral particles and the assembly and release of the virus. HMGB1 plays an important role in the antiviral process of the glycyrrhizin, and the HMGB1 is a very abundant nucleoprotein in a cell and can be released into the extracellular space as an important inflammatory medium to regulate the inflammatory reaction of the host. We found that the increase of the pro-inflammatory factor (IL-1, IL-6, IL-8, TNF-1) caused by PEDV infection and the increase in the pro-inflammatory factor by Poly (I: C) and the promotion of the infection of PEDV. Licorice is a competitive inhibitor of HMGB1. HMGB1 was found to play an important role in the increase of the pro-inflammatory factor under the condition of infection by the HMGB1 antibody and the expression of HMGB1 in the test and siRNA. The HMGB1 can be combined with a receptor such as TLR4 and RAGE, so that the cell is treated with a TLR4 inhibitor and a siRNA of RAGE, and it is found that the TLR4 inhibitor and siRAGE can reduce the increase in the proinflammatory factor and the infection of the virus, and that the mutant (PCA-C45S, PCA-C106S, PCA-C45106S) overexpressing the HMGB1 may reduce the increase in the proinflammatory factor and the infection of the PEDV. In addition, we have shown that the combination of HMGB1 with TLR4 and RAGE can activate the P38MAPK to induce the increase of the pro-inflammatory factor, which is beneficial to the infection of the PEDV. In conclusion, after the combination of the licorice and the extracellular HMGB1, the combination with the TLR4 and the RAGE receptor is reduced, so that the activation of the P38MAPK is reduced, and the proinflammatory factor is reduced to inhibit the proliferation of the PEDV. The sulfate-like heparin is the adsorption factor of the Vero cells infected with the porcine epidemic diarrhea virus, and the sulfate-like heparin is used as the adsorption factor of the virus. In this study, it was demonstrated that PEDV was used to adsorb Vero cells using heparan sulfate. Western blot analysis, fluorescence quantitative PCR and plaque formation assay revealed that the ability of the co-infected cells to be pre-treated by heparin was inhibited; while the cells were pretreated with heparin, the infection of the PEDV was not inhibited. Second, we found that the susceptibility of the cells to the PEDV decreased after the heparin enzyme I was treated to remove the sulfate-like heparin. In addition, we have shown that the inhibition of the sodium chlorate-treated cells of the sulfate-like heparin biosynthesis can effectively inhibit the infection of the PEDV. We also examined the effect of two heparin analogs of varying degrees of sulfation on the PEDV infection, which indicated that the N-desulphated heparin (not N-ethionized O-desulphonated heparin) pretreated the virus to reduce the ability of the virus to infect cells. In addition, heparin can inhibit the replication of PEDV and the level of HMGB1 mRNA. In conclusion, the study shows that the sulfate-like heparin is an adsorption factor for the Vero cells infected with PEDV. The PEDV N protein is an important pro-inflammatory mediator by interacting with C/ EBP-1 to regulate the transcription of HMGB1 and to release HMGB1, and to promote the occurrence of a variety of inflammatory diseases. The infection of the PEDV and the envelope protein of the PEDV were detected by SIRT1, histone B-transferase and NF-B-B to regulate the ethylation of HMGB1. Overexpression of the PEDVN protein can promote the ethylation and release of HMGB1. The CHIP test shows that the PEDVN can interact with the DNA of the HMGB1 promoter region. The double luciferase reporter gene test demonstrated that the transcription of the PEDVN protein-mediated HMGB1 was related to the transcription factor C/ EBP binding motif. The interaction of the capsid protein of the virus with the C/ EBP-1 was further shown by the Co-IP test to regulate the transcription of the HMGB1 gene. In conclusion, PEDV infection can cause the ethylation and release of HMGB1, and is mainly the function of the PEDV N protein. Our results provide a new perspective for the development of PEDV and provide a theoretical basis for the development of therapeutic methods and drugs targeting HMGB1 as a target. The release of HMGB1 caused by the DNA damage of the porcine epidemic diarrhea virus is widely studied by the DNA damage reaction activated by the DNA virus. However, there are few studies on the reaction of DNA damage caused by the RNA virus. Therefore, by western blot and indirect immunofluorescence, the phosphorylation of H2AX was rapidly activated by PEDV, and the expression of PEDVN protein was increased by HY. In addition, the DNA-PK inhibitor (NU7441) and the ATM inhibitor (KU-55933) have been shown to inhibit the release of HMGB1 and the infection of the PEDV by western blot, qRT-PCR and plaque formation test, and the effect of the inhibitor of DNA-PK is better. However, the ATR inhibitor (VE-821) has no effect on the PEDV infection. In addition, PEDV is responsible for the activation of PARP1, and the inhibitor 3-AB of PARP1 inhibits the release of HMGB1 and the infection of the PEDV. The activity of PARP1 was inhibited by the DNA-PK inhibitor (NU7441) and the ATM inhibitor (KU-55933), indicating that the PEDV primarily activates the proliferation of PARP1 and PEDV by inducing DNA-PK-dependent DNA damage and partial ATM-dependent DNA damage. The increase of ROS caused by PEDV was demonstrated by flow cytometry, and the inhibition of DNA damage and the activation of PARP1 could reduce ROS. In conclusion, PEDV is responsible for the release of HMGB1 by DNA damage/ PARP1/ ROS to facilitate its own replication.
【學位授予單位】：南京農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S858.28
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本文編號：2488193