Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/98197
標題: 探討TNFAIP2在H1N1 A型流感病毒感染中所扮演的角色
Characterization of the role of TNFAIP2 in influenza A H1N1 virus infection
作者: 李侑恩
Yu-En Li
關鍵字: 腺瘤壞死因子誘發蛋白質第二型(TNFAIP2)
A型流感病毒
NF-κB
AKT
CRISPR/Cas9
Tumor necrosis factor alpha-induced protein type 2
Influenza A virus
NF-κB
AKT
CRISPR/Cas9
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摘要: 腫瘤壞死因子誘發蛋白質第二型 (Tumor necrosis factor alpha induced protein type 2, TNFAIP2) 最初是選殖自受腫瘤壞死因子刺激的人類臍帶內皮細胞,可能參與血管新生及發炎反應。TNFAIP2的基因表現可透過NF-κB轉錄因子的活化而大量增加,而NF-κB可活化宿主免疫反應以對抗微生物感染,亦可能正向調控微生物本身在宿主細胞中的複製。有文獻指出,有些病毒在感染細胞後會促使TNFAIP2大量表現,然而及其分子功能上的意義還不清楚。已知H1N1 A型流感病毒在感染細胞後會活化NF-κB及AKT所參與之多種細胞訊號途徑,因此本研究將利用A549人類肺腺癌上皮細胞株,探討TNFAIP2在A型流感病毒H1N1 (PR8)感染中所扮演的角色。西方墨點實驗的結果顯示,在流感病毒感染後12和24小時,A549細胞中可偵測到病毒NS1蛋白的表現、NF-κB的活化以及AKT的磷酸化,然而細胞中TNFAIP2的表現量卻明顯受到抑制。我們以siRNA降低A549細胞中TNFAIP2的表現量後感染病毒,與對照組相比,病毒NS1蛋白的表現量在TNFAIP2表現量降低之A549細胞中較高,且病毒引起的NF-κB活化程度更為明顯。我們接著利用CRISPR/Cas9基因編輯技術剔除A549細胞的TNFAIP2基因,該細胞在病毒感染後12和24小時,病毒NS1和NP蛋白的表現量,以及NF-κB 和AKT的活化程度皆明顯增加。為了能進一步釐清TNFAIP2在流感病毒感染中的角色,我們構築了一系列的TNFAIP2表現載體,並以西方墨點及免疫螢光染色確認其在細胞中的表現,結果顯示截切TNFAIP2的 N端序列,會影響TNFAIP2在A549細胞中的分佈位置。綜合上述結果可推測TNFAIP2在流感病毒感染過程中,可能參與抑制病毒感染的宿主反應,未來還需進行更多實驗以進一步釐清TNFAIP2的角色。
Tumor necrosis factor alpha (TNFα)-induced protein type 2 (TNFAIP2) was originally identified as an inducible gene in TNFα-treated human endothelial cells and thus likely participates in angiogenesis and pro-inflammatory responses. TNFAIP2 expression can be transcriptionally upregulated via activation of the transcription factor NF-κB, which is known to mediate both pro- and antiviral functions. Infection of some viruses has been shown to induce TNFAIP2 expression; however, the functional influence remains largely unknown. In this study, the role of TNFAIP2 in the interplay between influenza A virus (IAV) H1N1 infection and cellular response was characterized in IAV-permissive A549 cells. Infection of A549 cells with IAV H1N1 led to expression of IAV-encoded NS1 protein at 12 and 24 hours post infection, in conjunction with activation of NF-κB and phosphorylation of AKT; however, TNFAIP2 expression was reduced by IAV infection. Depletion of TNFAIP2 expression by siRNA promoted viral NS1 expression and IAV H1N1-induced NF-κB activation. Furthermore, gene knockout of TNFAIP2 using the CRISPR/Cas9 system enhanced expression of viral NS1 and NP respectively at 12 and 24 hours post infection, in conjunction with enhanced activation of NF-κB and AKT in A549 cells. Finally, expression vectors for various truncated forms of TNFAIP2 were constructed, and their expression patterns were verified by western blotting and immunofluorescence staining. As a result, deletion of the N-terminus of TNFAIP2 led to its altered intracellular localization. Collectively, the present data suggest that TNFAIP2 may play an inhibitory role in early phase of IAV H1N1 infection. Further investigations are needed to better elucidate underlying mechanisms.
URI: http://hdl.handle.net/11455/98197
文章公開時間: 2018-08-16
Appears in Collections:微生物暨公共衛生學研究所

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