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  • 學位論文

雙股RNA病毒經由腫瘤壞死因子α (TNFα) 調控宿主細胞表現細胞素與細胞凋亡之途徑

Tumor Necrosis Factor α regulates cell apoptosis and cytokine expression in the pathogenesis of birnavirus

指導教授 : 吳金洌

摘要


感染性胰臟壞死病毒(Infectious Pancreatic Necrosis Virus, IPNV)為兩段雙股核醣核酸病毒科(Birnaviridae)的魚類病毒,對漁撈及水產養殖上皆造成重大損失。過去研究發現,IPNV在感染魚類細胞後,可造成宿主細胞發生細胞凋亡(apoptosis)與細胞壞死(necrosis)的現象,此外抗細胞凋亡基因Mcl-1表現量下降而促細胞凋亡基因Bad表現量增加,並經由tyrosine kinase調控途徑活化下游的caspase-8與caspase-3,使被感染細胞走向細胞凋亡,但IPNV藉由何種機制調控並決定被感染宿主細胞走向細胞凋亡或細胞壞死的機制仍然並不清楚。 以斑馬魚微矩陣晶片組(oligo microarray)分析在斑馬魚胚胎細胞(ZF4)被IPNV感染6、12與24小時後,在免疫與細胞凋亡上轉錄表現改變的相關基因組,結果顯示腫瘤壞死因子α (TNFα)與多數促細胞凋亡(pro-apoptotic)的Bcl-2家族基因表現量在病毒感染初期即開始增加,進一步以腫瘤壞死因子α專一性的小片段干擾核醣核酸(siRNA)或其抑制劑tyrphostin AG-126處理細胞再感染病毒後,宿主細胞的死亡率與細胞凋亡的情形皆明顯下降,同時Bcl-2家族促細胞凋亡基因Bad與Bid的表現量以及caspase-3, -8與-9的活化情形都受到明顯的抑制。此外以腫瘤壞死因子α或促細胞壞死基因RIP-1專一性的siRNA及其抑制劑tyrphostin AG-126處理後,宿主細胞壞死比例與活性氧化物(ROS)的生成量也有明顯的下降情形。因此由研究結果可知,魚類細胞株在遭受感染性胰臟壞死病毒感染時,藉由活化腫瘤壞死因子α之途徑啟動細胞凋亡與壞死之機制以抵抗病毒之有效入侵。 以定量反轉錄聚合酶連鎖反應(quantitative real-time PCR)分析在IPNV感染後的斑馬魚胚胎細胞,在免疫反應及相關調節因子轉錄表現量上的改變,結果顯示包括ifna, ifng, mx, irf1, irf2, irf4, tnfa, tnfb, il-1b, il-15, il-26, ccl4與mmp家族基因被誘導表現,此外免疫調節因子cebpb, junb, nfkb, stat1, stat4與stat5表現量亦增加。進一步以軟體(Pathway Studio software)分析發現,腫瘤壞死因子α影響多數下游免疫與細胞素基因在IPNV感染後的表現量,將被感染細胞以腫瘤壞死因子α的siRNA或其專一性抑制劑tyrphostin AG-126處理後,轉錄因子NF-κB的表現量受到抑制,進一步研究發現干擾素與部份細胞間素的表現受到腫瘤壞死因子α與轉錄因子NF-κB連鎖途徑調控。 我們以斑馬魚微小核醣核酸微矩陣晶片組(microRNA microarray)與定量PCR分析IPNV所感染宿主細胞的microRNA表現量後發現,miR-132、miR-146a與miR-155的轉錄表現量增加,而miR-125b的表現量則在感染後下降。過去的研究發現miR-125b可藉由攻擊腫瘤壞死因子α的3端未轉譯區域 (3’-UTR) 降低其表現量。研究結果指出魚類細胞以miR-125 mimic處理後再感染病毒,腫瘤壞死因子α的表現量較未處理之細胞低,此外被感染後的宿主細胞凋亡與壞死的比例亦較未處理細胞為低。以miR-125b mimic處理後的細胞感染IPNV後,caspase的活化情形與活性氧化物的累積也受到抑制。因此我們的結果顯示miR-125b在病毒感染的宿主細胞中可作為腫瘤壞死因子α途徑引發細胞凋亡與壞死的負向調控者。 感染性胰臟壞死病毒之致病機制上,腫瘤壞死因子α的角色與功能至為重要,除了可調控宿主細胞的細胞凋亡與壞死之外,亦可藉由調控下游轉錄因子NF-κB增加免疫相關細胞間素或干擾素的表現,可在雙股RNA病毒的研究與防治上可提供更為有效的策略與應用。

並列摘要


The infectious pancreatic necrosis virus (IPNV) belongs to the Birnaviridae family of viruses and causes acute contagious diseases in a number of economically important freshwater and marine fish. Previous studies have shown that IPNV induces both atypical apoptosis and secondary necrosis in fish cells. The expression of the survival factor Mcl-1 has been shown to be down-regulated and that of the pro-apoptotic bcl-2 family gene Bad has been shown to be up-regulated by IPNV. IPNV infection can trigger the tyrosine kinase-mediated death pathway and cause the activation of caspase-8 and -3 in virus-infected cells. IPNV can induce Bad-mediated apoptosis followed by secondary necrosis in fish cells, but it is not known how these two types of cell death are regulated by IPNV. Using DNA microarray and quantitative RT-PCR analyses, two major subsets of differentially expressed genes were characterized, including the innate immune response gene TNFα and the pro-apoptotic genes Bad and Bid. In the early replication stage, we observed that the pro-inflammatory cytokine TNFα underwent a rapid six-fold induction. Then during the early-middle replication stages, the TNFα level was eight-fold higher, and the pro-apoptotic Bcl-2 family members Bad and Bid were also up-regulated. Specific inhibitors of TNFα expression (AG-126 or TNFα-specific siRNA) were used to block apoptotic and necrotic death signaling during the early or early-middle stages of IPNV infection. Inhibition of TNFα expression dramatically reduced the activity of the Bad/Bid-mediated apoptotic and Rip1/ROS-mediated necrotic cell death pathways and rescued host cell viability. The Rip1/ROS-mediated secondary necrotic pathway appeared to be reduced in IPNV-infected fish cells during the middle-late stage of infection. We also infected zebrafish embryonic (ZF4) cells with IPNV and analyzed the gene expression patterns of normal and infected cells using quantitative real-time PCR. We identified a number of immune response genes, including ifna, ifng, mx, irf1, irf2, irf4, tnfa, tnfb, il-1b, il-15, il-26, ccl4 and mmp family genes, that are induced after viral infection. Transcriptional regulators, including cebpb, junb, nfkb and stat1, stat4 and stat5, were also up-regulated in IPNV-infected cells. In addition, we used Pathway Studio software to identify TNFα as the factor with the greatest downstream influence among these altered genes. Treating virus-infected cells with an siRNA targeting TNFα inhibited NF-κB expression. To further interrupt the TNFα/NF-κB-mediated pathway, the expression levels of cytokines and metalloproteinases were inhibited in IPNV-infected cells. Using microRNA array and real-time quantitative PCR assays, the expression patterns of microRNAs in IPNV-infected fish cells were characterized during different replication stages of IPNV. We found that the gene transcription levels of miR-132, miR-146a and miR-155 were up-regulated, and miR-125b was down-regulated. Previous studies have shown that the 3’-untranslated regions of TNFα transcripts can be targeted by miR-125b. A miR-125b mimic or a TNFα-specific siRNA was able to down-regulate the expression of TNFα in IPNV-infected cells. Following miR-125b mimic treatment, the viability of IPNV-infected cells was increased. The percentages of apoptotic and necrotic IPNV-infected ZF4 cells that were pretreated with a miR-125b mimic or a TNFα-specific siRNA were decreased, as shown by fluorescence images of Annexin V-fluorescein and PI staining. The activation of caspase-3, -8, and -9 and the formation of ROS were inhibited following miR-125b mimic or TNFα-specific siRNA treatment of ZF4 cells infected with IPNV. Therefore, this work indicates that miR-125b is suppressed in response to IPNV and acts as a negative regulator of TNFα-mediated apoptosis and secondary necrosis induced by IPNV. Taken together, our results indicate that IPNV triggers two death pathways via upstream induction of the pro-inflammatory cytokine TNFα and that the expression of cytokines and metalloproteinases might be initiated through the TNFα/NF-κB-mediated pathway. TNFα plays important roles in cell death and immune responses during IPNV infection. These results may provide new insights into the pathogenesis of RNA viruses.

並列關鍵字

IPNV zebrafish TNFα apoptosis necrosis microRNA

參考文獻


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