本文選題：登革病毒　切入點：抗體依賴性增強感染　出處：《北京協和醫(yī)學院》2015年博士論文

【摘要】：登革熱病毒(Dengue virus, DENV)是一種蚊媒病毒,在超過100多個國家中,尤其是亞洲和拉丁美洲,引起了巨大的公共衛(wèi)生問題。據估計,每年有超過5千萬至1億人感染登革熱病毒。隨著其宿主埃及伊蚊和白紋伊蚊棲息地的擴張,全球面臨越來越嚴重的登革感染風險。四型登革病毒中每一型單獨感染均能導致一系列程度不一的臨床癥狀,從自限性的登革熱(DF)至危及生命的登革出血熱(DHF)或登革休克綜合癥(DSS)等重癥形式。基于臨床觀察,二次異型感染或登革患者胎兒隨年齡增長均顯著增加重癥感染幾率,抗體依賴增強假說(ADE)被提出以解釋登革病毒重癥感染病理機制。該理論認為,在二次異型登革病毒感染中預先存在的交叉抗體能夠與病毒結合,通過抗體與靶細胞表面Fc受體的相互作用促進病毒對單核細胞,巨噬細胞,以及成熟DC細胞的感染。目前研究認為DENV-ADE能夠通過抑制Ⅰ型干擾素產生及效應進而促進病毒增殖；其中抗炎癥細胞因子,如IL-10等對干擾素途徑的抑制作用在DENV-ADE感染中扮演的重要角色。然而有研究證明DENV-ADE感染的人初始單核細胞并不抑制干擾素或IL-10表達。這些研究顯示DENV-ADE感染Fc受體陽性細胞存在一種更為普遍的機制,且不依賴于上調IL-10對一型干擾素抑制過程。在本研究中,我們使用Ⅰ型干擾素缺陷細胞K562建立了DENV3抗體依賴增感染體外模型,并體現出內部增強感染形式。通過檢測抗病毒基因NOS2表達水平,發(fā)現DENV-ADE感染細胞中降低的NOS2表達及NO活性分子釋放導致了病毒胞內復制增加。進一步分析NOS2基因上游調控途徑,發(fā)現DENV-ADE感染中下調的NOS2基因表達源于固有免疫RIG-I/MDA-5—NF-κB—IRF-1信號途徑抑制。過表達RIG-I和/或MDA-5能夠協同促進NF-κB活化及NOS2表達。為驗證免疫抑制因子IL-10在DENV-ADE固有免疫抑制中的作用,我們檢測了IL-10/IL-6—SOCS3通路,發(fā)現DENV-ADE感染K562細胞未顯著上調IL-10, IL-6, SOCS3表達。利用CRISP/CAS9技術獲得IL-10敲出的K562細胞進行DENV-ADE模型表明,IL-10并不影響DENV-ADE感染。由于細胞自噬過程在DENV復制中扮演重要作用,我們比較了DENV直接感染與DENV-ADE感染中自噬程度差異。研究發(fā)現,DENV-ADE感染K562細胞誘導更高水平的自噬體形成及自噬相關蛋白ATG5, ATG12表達。通過藥物促進或抑制細胞自噬進程能夠劑量依賴性地促進或抑制胞內病毒復制,且完全敲除自噬相關蛋白ATG5顯著抑制胞內病毒復制。結果表明,DENV-ADE感染通過誘導更強的自噬進而促進胞內病毒增殖。此外,過表達自噬蛋白ATG5能夠通過抑制NF-κB活化及NOS2表達從而促進病毒增殖。本研究發(fā)現DENV-ADE感染中病毒復制增強是由RIG-I/MDA-5—NF-κB— IRF-1—NOS2固有免疫途徑抑制導致的,且該抑制過程不依賴于IL-10—SOCS3對干擾素途徑的抑制。此外DENV-ADE感染能夠通過上調細胞自噬進程,抑制固有免疫途徑,從而促進病毒增殖。該研究將有助于加深我們對DENV-ADE感染機制的理解,并為重癥登革患者治療提供了理論支持和潛在靶點,同時也表明自噬抑制劑在治療DENV-ADE引起的重癥登革熱疾病,以及抑制病毒胞內復制方面具有潛在的應用價值。
[Abstract]:Dengue virus (Dengue virus DENV) is a mosquito borne virus, in more than more than 100 countries, especially in Asia and Latin America, causing a major public health problem. It is estimated that there are more than 50 million to 100 million people infected with dengue virus each year. With its host Egypt and Iraq mosquito Aedes albopictus habitat expansion the world, facing increasingly serious risk of dengue infections. Dengue virus type four in each type of single infection can cause a series of clinical symptoms in different degree, from self limited dengue fever (DF) to life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) in form. Based on clinical observation, two patients with dengue infection or abnormal fetal age were significantly increased in severe infection, antibody dependent enhancement hypothesis (ADE) is proposed to explain the dengue virus infection. The pathological mechanism of theory in the two different Dengue virus infection cross pre-existing antibodies can bind to the virus and antibody through interaction with the target cell surface Fc receptor on monocyte macrophage to promote viral infection, and, mature DC cells. The present study suggests that DENV-ADE can inhibit type I interferon production and effect and promote the proliferation of virus; anti inflammation cytokines, such as inhibition of IL-10 on interferon pathway in DENV-ADE infection plays important role. However, studies have shown that the initial DENV-ADE infection of monocytes did not inhibit interferon or IL-10 expression. These studies showed the presence of a common infection mechanism of DENV-ADE Fc receptor positive cells, and does not depend on the up regulation of IL-10 inhibit the process of a type of interferon. In this study, we use the type I interferon deficient cells K562 established DENV3 antibody dependent enhancement of infection. Outside the model, and reflect the internal form of infection. Enhanced expression levels by detection of antiviral gene NOS2, found that the virus has led to increased release of intracellular replication molecule NOS2 expression cells decreased and the activity of NO DENV-ADE infection. Further analysis of NOS2 gene upstream regulatory pathways, found that DENV-ADE infection in down-regulation of NOS2 gene expression in innate immune RIG-I/MDA-5 NF- B - kappa IRF-1 signaling pathway inhibition. Overexpression of RIG-I and / or MDA-5 can synergistically promote NOS2 activation and expression of NF- kappa B. In order to verify the immunosuppressive factor IL-10 in innate immune suppression in DENV-ADE, we examined the IL-10/IL-6 SOCS3 pathway, found DENV-ADE infected K562 cells did not significantly up-regulated the expression of IL-10, IL-6, SOCS3 expression get out of IL-10. By using the technology of CRISP/CAS9 K562 cell DENV-ADE model showed that IL-10 did not affect DENV-ADE infection due to autophagy in DENV. Play an important role in replication, we compared the autophagy of DENV direct infection and DENV-ADE infection in difference. The study found that autophagy induced higher levels of DENV-ADE infected K562 cells and the formation of autophagy related protein ATG5, ATG12 expression. The drugs promote or inhibit autophagy process could dose dependently promote or inhibit virus replication in cells completely, and knockdown of autophagy related protein ATG5 significantly inhibited the virus replication in cells. The results showed that DENV-ADE infection induced by stronger autophagy and promote the proliferation of virus in the cells. Moreover, overexpression of autophagy protein ATG5 can inhibit NF- B activation and NOS2 expression to promote proliferation of virus. This study found that DENV-ADE infection enhanced virus replication is caused by RIG-I/MDA-5 - NF- - IRF-1 - NOS2 - B innate immune pathways inhibition, and the inhibition process does not depend on the IL-10 - SOCS3 of interferon The way to inhibit DENV-ADE infection. In addition to the upregulation of cell autophagy process, inhibition of innate immune pathways, thereby promoting proliferation of virus. The study will help to deepen our understanding of the mechanism of DENV-ADE infection, and to provide theoretical support and a potential target for treatment of patients with severe leather also shows that, in the treatment of DENV-ADE induced autophagy inhibitors the severe dengue disease has potential application value and inhibit viral intracellular replication.

【學位授予單位】：北京協和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R392

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