【摘要】：固有免疫(innate immunity)是機體抵抗病毒的第一道防線。當病毒入侵機體后,即被模式識別受體(pattern-recognition receptors,PRRs)識別,快速誘導固有免疫應答,產(chǎn)生大量的Ⅰ型干擾素(interferon,IFN)和促炎因子。Ⅰ型干擾素可以與胞膜上相應的受體結合,誘導一系列抗病毒蛋白高表達,進而抑制病毒的復制和感染,發(fā)揮強有力的抗病毒效應[1,2]。因此,Ⅰ型干擾素的表徶調控一直是抗病毒免疫研究領域的熱點。自噬是一種發(fā)生在真核細胞內并且高度保守的自我穩(wěn)態(tài)機制,對細胞抵抗外部刺激有非常重要的意義。病毒感染可以引起自噬的產(chǎn)生,但是目前關于自噬在病毒感染過程中所起的作用的研究很少,機制尚不明確。所以我們對自噬在抗病毒固有免疫應答中的作用機制進行了探究。我們構建了自噬相關基因7(Autophagy related gene 7,ATG7)在髓系細胞中特異性敲除的小鼠(Atg7f/f Lysm-Cre)。用VSV-GFP病毒感染小鼠的腹腔巨噬細胞,發(fā)現(xiàn)自噬缺陷之后小鼠的腹腔巨噬細胞對病毒的清除率增強,具有更強的抗感染能力。隨后,我們給小鼠腹腔注射VSV病毒觀察小鼠的存活率,得到了和體外實驗一致的結果,自噬缺陷組小鼠的存活率明顯升高,肺部組織炎性浸潤增強,巨噬細胞、中性粒細胞富集明顯增多,病毒復制減少,小鼠抗病毒能力明顯增強。因為Ⅰ型干擾素在抗病毒固有免疫中發(fā)揮著重要作用,所以我們接著探究了自噬缺陷引起的這種抗病毒效應是否是因為影響了Ⅰ型干擾素的產(chǎn)生。所以我們用病毒感染小鼠的腹腔巨噬細胞,檢測了對照組和自噬敲除組小鼠腹腔巨噬細胞Ⅰ型干擾素mRNA的表徶情況,發(fā)現(xiàn)病毒感染之后自噬缺陷組的Ⅰ型干擾素mRNA表達明顯增強。證實了自噬缺陷確實影響了Ⅰ型干擾素的合成,從而增強了抗病毒效應。VSV病毒是一種dsRNA病毒,主要通過活化TLR3信號通路和RIG-Ⅰ信號通路來引起Ⅰ型干擾素的產(chǎn)生,隨后我們對RNA病毒活化的信號通路進行了篩選和驗證。我們用免疫印跡實驗對不同信號通路主要的抗病毒蛋白的降解情況進行了檢測,發(fā)現(xiàn)自噬缺陷之后TBK1的磷酸化水平增加,IRF3總蛋白和磷酸化水平都增加,而NF-κB的降解沒有差異。同時還檢測了 IRF3 mRNA水平,結果顯示自噬缺陷后IRF3的合成亦增多。綜上所述,我們發(fā)現(xiàn)自噬缺陷可以引起IRF3活化形式增多,促進Ⅰ型干擾素的產(chǎn)生,進而增強機體抗病毒能力。本研究揭示了自噬在抗病毒固有免疫中重要的負調控作用,為抗病毒感染免疫的調控機制提供新的觀點,為病毒感染的臨床治療方法提供指導,為藥物新靶點的設計提供了思路。
[Abstract]:Innate immune (innate immunity) is the body's first line of defense against the virus. When the virus invades the body, it is recognized by the pattern recognition receptor (pattern-recognition receptors,PRRs), which can quickly induce the innate immune response and produce a large number of interferon type I (interferon,). IFN) and pro-inflammatory factor.Interferon I can bind to the corresponding receptors on the cell membrane, induce a series of high expression of antiviral proteins, and then inhibit the replication and infection of the virus, and play a powerful antiviral effect [1t2]. Therefore, epidermal regulation of interferon type I has been a hot topic in the field of antiviral immunity. Autophagy is a highly conserved self-homeostasis mechanism occurring in eukaryotic cells, which plays an important role in the resistance of cells to external stimuli. Virus infection can induce autophagy, but there is little research on the role of autophagy in the process of virus infection, and the mechanism is unclear. So we explored the mechanism of autophagy in anti-viral innate immune response. We constructed a mouse (Atg7f/f Lysm-Cre) with specific knockout of autophagy associated gene 7 (Autophagy related gene 7 (ATG7) in myeloid cells. The peritoneal macrophages of mice infected with VSV-GFP virus were found to have stronger anti-infection ability and the clearance rate of peritoneal macrophages was enhanced after autophagy deficiency. Then, we injected VSV virus intraperitoneally into mice to observe the survival rate of mice. The results were consistent with in vitro experiment. The survival rate of mice in autophagy deficiency group was significantly increased, the inflammatory infiltration of lung tissue was enhanced, and macrophages were also observed. The concentration of neutrophil increased, the replication of virus decreased, and the anti-viral ability of mice increased obviously. Because interferon type I plays an important role in the innate antiviral immunity, we then explore whether the antiviral effect of autophagy deficiency is due to the influence of interferon type I production. So we used the virus to infect the peritoneal macrophages of mice, and we detected the expression of interferon type I mRNA in the peritoneal macrophages of the control group and the mice in the autophagy knockout group. It was found that the expression of interferon type I mRNA in autophagy deficiency group was significantly increased after viral infection. It is confirmed that autophagy deficiency does affect the synthesis of interferon type 鈪，

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