Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/66424
標題: 克沙奇病毒B3型誘發老鼠神經膠細胞產生一氧化氮和細胞激素之研究
Nitric oxide and Cytokine Production by Rat Glial cell Stimulated with Coxsackievirus B3 in vitro
作者: 楊境評
Ching-Ping, Yang
關鍵字: Coxsackievirus group B3;克沙奇病毒B3型;nitric oxide;glial cell;MAPK pathway;NF-κB;MAPK路徑;一氧化氮;神經膠細胞
出版社: 獸醫微生物學研究所
摘要: 
中文摘要
克沙奇病毒B3型是屬於微小RNA病毒科家族中的腸病毒屬。新生兒容易受到克沙奇病毒感染中樞神經系統而造成腦膜炎、腦炎,長時間的感染會造成神經性的退化。目前,克沙奇病毒造成神經性疾病的機轉仍然是不了解的。當神經細胞受到外來刺激時,會產生誘發型一氧化氮合成酶來製造大量的一氧化氮,這是神經細胞產生發炎反應的重要機轉之一,而一氧化氮在神經系統也具有神經保護及神經傷害的功能。在過去的研究中發現在活體動物實驗上,受到某些病毒例如單純疱疹病毒第一型、Junin virus感染老鼠同樣在腦部有發炎的現象,包括了免疫性細胞的滲入,發炎因子一氧化氮、腫瘤壞死因子-α、介白素-1β、干擾素-γ的產生。我們初步的實驗中發現,當神經元/神經膠混合細胞受到克沙奇病毒感染,會產生大量的一氧化氮,這現象和活體動物實驗上所獲得的結果是一致的。一般而言,腦部中一氧化氮往往是透過活化神經膠細胞所產生。因此,本論文探討在初代培養的混合神經膠細胞中,克沙奇病毒感染誘發一氧化氮生成的主要細胞種類及作用機轉。利用所培養不同的神經細胞進一步分析發現,除了神經元/神經膠混合細胞外,混合神經膠細胞及微神經膠細胞亦會產生一氧化氮,但是,星狀神經膠細胞卻不會產生一氧化氮。另外,我們從神經膠細胞的形態改變、細胞激素產生情形去探討細胞活化之情形,發現克沙奇病毒分別感染混合神經膠細胞、微神經膠細胞、星狀神經膠細胞後會誘發細胞激素產生,在細胞形態上,只在微神經膠細胞發現有明顯的改變,星狀神經膠細胞則無明顯的形態改變。總合結果我們發現克沙奇病毒感染會活化神經膠細胞,特別是微神經膠細胞,而活化的微神經膠細胞會產生一氧化氮。進一步探討克沙奇病毒誘發一氧化氮生成之作用機轉。以紫外光去活化、熱去活化的克沙奇病毒感染細胞都會降低一氧化氮的產量,顯示病毒的複製在神經膠細胞產生一氧化氮的過程扮演重要的角色。在細胞內訊息傳遞機制方面,經由西方點墨法分析發現,克沙奇病毒感染的細胞,ERK和JNK磷酸化的程度會上升,若加入MAPK路徑抑制劑U0126、SP600125,則會降低一氧化氮的生成量,同時也降低發炎性細胞激素表現。顯示細胞內ERK、JNK路徑對於克沙奇病毒所造成一氧化氮合成酶的活化和細胞激素的產生佔有重要的角色。在轉錄的層次上,我們也發現病毒感染後的神經細胞,轉錄因子NF-κB會有活化的現象,而加入抑制劑PDTC能明顯降低一氧化氮、IL-6和RANTES。顯示NF-κB對於克沙奇病毒B3型感染神經細胞所誘發一氧化氮、L-6和RANTES產生伴演重要的功能。總結來說,我們發現克沙奇病毒感染神經膠細胞後會活化細胞來產生一氧化氮和發炎性的細胞激素,而活化的路徑中, MAPK路徑、NF-κB佔有重要的角色來調控一氧化氮和細胞激素的產生。

Abstract
Coxsackievirus group B3 (CVB3) within the genus enterovirus, which is a member of the Picornavirus family. Neonates are particularly susceptible to coxsackievirus infections of the central nervous system (CNS), which can cause meningitis, encephalitis, and long-term neurological deficits. The mechanisms by which CVB3 causes neurological diseases are not fully understood. The activation of inducible nitric oxide synthase (iNOS) and consequent production of nitric oxide (NO) in neural cells play important roles in the regulation of brain inflammation. NO exerts two opposite biological functions in the CNS including neuroprotective and neurotoxic effects. In animal study, the infection with HSV-1 or Junin virus in mice is characterized at the pathologic level by significant infiltration of inflammatory cells and the production of pro-inflammatory factors such as NO, TNF-alpha, IL-1beta and IFN-gamma. In the beginning of our study, we found that CVB3-infected neuron/glia cells, consisting of neuron, astrocyte, and microglia, produced NO. The generation of NO in in vitro neural cells after CVB3 infection is mimicked the in vivo animal study showing the activation of nitric oxide synthase activity in brain tissues after virus inoculation. Generally, large amounts of NO are produced by activated glial cells. Therefore, we assessed whether glial cells played roles in the induction of NO synthesis after CVB3 infection and tried to elucidate the underlying mechanisms. In addition to neuron/glia, mixed glia and microglia were able to produce NO after CVB3 infection. However, astrocytes were unable to release NO. Next, we examined whether CVB3 infection stimulated glial activation by the following assays including morphological change and cytokine expression. Obvious change in morphology was only observed in microglia after CVB3 infection. CVB3-infected mixed glia, astrocyte, or microglia all released various cytokines with variable efficiency. In short conclusion, CVB3 infection activated glial cells, especially microglia, and the activated microglia could produce NO. The mechanisms of CVB3-induced NO production were further characterized. Both UV and heat-inactivated CVB3 lost their ability to stimulate glial cells release NO. These findings imply that the event of viral amplification is essential to NO production in CVB3-infected glial cells. Regarging the intracellular signaling pathways, CVB3 infection markedly increased the phosphorylation level of ERK and JNK in glial cells. We found that treatment of glial cells with ERK/MEK inhibitor U0126 and JNK inhibitor SP600125 reduced the production of NO and cytokines. Based on the western blot analysis, the importance of ERK and JNK signaling pathway in the induction of iNOS and cytokine production after CVB3 infection in glial cells was appreciated. CVB3 infection increased the DNA binding ability of NF-κB, and the reduction of NO and cytokine production by the treatment of NF-κB inhibitor, PDTC, indicated the requirement of NF-κB activate in CVB3-metiated gene induction. Our current study provides evidence showing the underlying signaling pathways for NO and cytokine expression in CVB3-infected glial cells, which will shed light on the regulation of brain inflammation.
URI: http://hdl.handle.net/11455/66424
Appears in Collections:微生物暨公共衛生學研究所

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