請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/20082
標題: 日本腦炎病毒感染誘發神經膠細胞產生一氧化氮之研究
Japanese encephalitis virus stimulates nitric oxide production by glial cells
作者: 陳建宏
Chen, Jian-Hong
關鍵字: Japanese encephalitis virus
日本腦炎病毒
Microglia
Nitric oxide
Inducible nitric oxide synthase
微神經膠細胞
一氧化氮
誘發性一氧化氮合成酶
出版社: 生物醫學研究所
摘要: 當神經細胞受到外來刺激時,會產生誘發性一氧化氮合成酶 (iNOS) 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日本腦炎的患者,腦部會有巨噬細胞、少量的T細胞及B細胞的滲入及發炎反應的生成。另外組織切片染色結果也發現日本腦炎病毒主要感染神經元細胞 (Neuron)。在活體動物實驗上,日本腦炎病毒感染的老鼠也同樣有腦部發炎的現象,包括了免疫性細胞的滲入,發炎因子 [一氧化氮、腫瘤壞死因子阿法 (TNF-α)、介白素8 (IL-8)] 的產生,其中一氧化氮的產生會使得病毒生長量下降,老鼠的生存率增加。我們初步的實驗中發現,當神經元/神經膠混合細胞 (Neuron/glia) 受到日本腦炎病毒感染,會產生大量的一氧化氮,這現象和活體動物實驗所獲得的結果一致。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,本論文擬探討在初代培養的混合神經膠細胞 (Mixed glia) 中,日本腦炎病毒 (JEV) 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 (Microglia) 亦會產生一氧化氮,但是,神經元細胞及星狀神經膠細胞 (Astrocye) 卻不會產生一氧化氮。另外,我們從神經膠細胞的形態、細胞激素、細胞增生情形來探討細胞活化的情形。我們發現日本腦炎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。最後在細胞增生方面,只有混合神經膠細胞有增生現象,微神經膠細胞及星狀神經膠細胞則沒有增生現象。總合結果我們發現日本腦炎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。進一步探討日本腦炎病毒誘發一氧化氮生成之作用機轉。以紫外光去活化 (UV-inactivated)、熱去活化 (Heat-inactivated) 的日本腦炎病毒及處理2-AP後的感染細胞都會降低一氧化氮的產量,顯示病毒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,我們發現日本腦炎病毒感染過的細胞若加入MAPK路徑抑制劑U0126,則會降低一氧化氮的生成量,同時也降低誘發性一氧化氮合成酶的訊息RNA (mRNA) 及蛋白質表現。經由西方墨點法分析 (Western blot) 發現,日本腦炎病毒感染過的細胞內ERK磷酸化的程度會上升,當加入U0126後,會使ERK磷酸化程度下降。顯示細胞內ERK路徑對於日本腦炎病毒所造成誘發性一氧化氮合成酶的活化佔有重要的角色。另外,在轉錄的層次上,我們也發現病毒感染過後的神經細胞,會有轉錄因子 NF-κB 活化的現象,當加入 U0126 後則能夠降低 NF-κB 的活化程度。U0126降低NF-κB活化的情形也反應到誘發性一氧化氮合成酶訊息RNA及蛋白質表現的影響。顯示了,NF-κB在日本腦炎病毒感染神經細胞所誘發的誘發性一氧化氮合成酶轉錄機轉上的控制,扮演著極重要的功能。最後,探討日本腦炎病毒感染神經膠細胞所誘發一氧化氮的功能。在本篇的研究中發現,在感染日本腦炎病毒的細胞中加入SNP及SNAP來增加細胞內一氧化氮的產量,發現會抑制日本腦炎病毒RNA的合成,而加入L-NAME及PTIO來降低一氧化氮濃度,則會增加日本腦炎病毒RNA的合成,顯示一氧化氮會抑制日本腦炎病毒複製。另外我們也發現一氧化氮的多寡會調節日本腦炎病毒所誘發細胞激素的表現。總結來說,我們發現日本腦炎病毒感染神經膠細胞後會活化微神經膠細胞來產生一氧化氮,而活化的路徑中,病毒生長複製、ERK MAPK路徑、NF-κB都佔有重要的角色,而所產生的一氧化氮具有抑制日本腦炎病毒複製及調節細胞激素表現的功能。
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 central nervous system (CNS) including neuroprotective and neurotoxic effects. Clinically, inflammatory responses such as the infiltration of macrophages, small numbers of T cells and B cells as well as production of pro-inflammatory cytokines are detected in the brain, plasma or cerebrospinal fluids of Japanese encephalitis virus (JEV)-infected patients. The immunohistochemical studies reveal that JEV antigen was mainly localized in neurons. However, the role of glial cells in the replication or pathogenesis of JEV is limited. In animal study, the infection with JEV in mice is characterized at the pathologic level by significant infiltration of inflammatory cells and the production of pro-inflammatory factors. Among the measurable factors, NO exerts antiviral effect by inhibiting the growth of JEV and increasing the survival rate of infected mice. In the beginning of our study, we found that JEV-infected neuron/glia cells, consisting of neuron, astrocyte, and microglia, produced NO. The generation of NO in in vitro neural cells after JEV infection is mimicked the in vivo animal study activating nitric oxide synthase activity in brain tissues after JEV 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 JEV infection and elucidated the underlying molecular mechanisms. In addition to neuron/glia, mixed glia and microglia were able to produce NO after JEV infection. However, neurons and astrocytes were unable to release NO. Next, we examined whether JEV infection stimulated glial activation by the following assays including morphological change, cytokine expression, and cell proliferation. Obvious change in morphology was only observed in microglia after JEV infection. JEV-infected mixed glia, astrocyte, or microglia all released various cytokines with variable efficiency. JEV infection stimulated cell proliferation in mixed glia. Whereas, JEV-induced cell proliferation could not be found in both astrocyte and microglia cultures. In short conclusion, JEV infection activated glial cells, especially microglia, and the activated microglia could produce NO. The mechanisms of JEV-induced NO production were further characterized. Both UV-inactivated and heat-inactivated JEV lost their ability to stimulate glial cells release NO. PKR inhibitor 2-AP attenuated JEV-induced NO production in glial cells. These findings imply that viral amplification event is critical to NO production in JEV-infected glial cells. Regarding the intracellular signaling pathways, we found that treatment of glial cells with ERK/MEK inhibitor U0126 reduced the production of NO, expression of iNOS protein and mRNA level. Based on the western blot analysis, JEV infection markedly increased the phosphorylation level of ERK in glial cells and the increase was blocked by the treatment with U0126, indicating the importance of ERK signaling pathway in the induction of iNOS activity after JEV infection in glial cells. The activation of iNOS gene expression is mostly through the transcriptional control, especially via transcription factor NF-kB. JEV infection increased the DNA binding ability of NF-kB in glial cells and the increase was attenuated by the treatment with U0126. These results revealed that transcription factor NF-kB played an important role in the induction of iNOS in JEV-infected glial cells. Finally, we tried to elucidate the biological functions of released NO after JEV infection in glial cells. We hypothesized that NO might exert antiviral effect and the elevation of NO production after JEV infection could suppress JEV amplification and reduced JEV-induced cellular alterations. In this study, increased NO production was achieved by treatment of cells with organic NO donors such as SNP or SNAP. In contrast, the suppression of JEV-induced NO production was done by treating cells with NOS inhibitor L-NAME or NO scavenger PTIO. Treatment of JEV-infected glial cells with SNP and SNAP reduced viral amplification but, L-NAME and PTIO increased that. These four agents modulated JEV-induced cytokine production with distinct characteristics. Taken together, we found that microglia responded to JEV infection by release of NO. The event of viral amplification, ERK signaling pathways, and transcription factor NF-kB were critical for iNOS gene expression. Finally, viral infection induced NO production in glial cells and the consequent NO production could suppress JEV replication and modulate JEV-induced cytokine expression.
URI: http://hdl.handle.net/11455/20082
顯示於類別:生物醫學研究所

文件中的檔案:
沒有與此文件相關的檔案。


在 DSpace 系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。