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標題: 豬瘟病毒感染內皮細胞對一氧化氮的影響
The Effect of Classical Swine Fever Virus (CSFV) on Nitric Oxide (NO) in Porcine Aortic Endothelial Cells (PAECS)
作者: 王之仰
關鍵字: 基礎研究
Classic swine fever virus(CSFV)
nitric oxide
nitric oxide synthase
摘要: 猪瘟是一種在猪隻上常見的重要傳染病。具有很高的傳染率及死亡率。雖然目前大規模免疫計畫已被實施,但散發性的病例仍造成畜牧業重大的經濟損失。猪瘟病毒屬於黃病毒科的瘟疫病毒屬。在急性或亞急性病例中,激烈的炎症及免疫反應與過度活化的凝血活性會引起身體組織及臟器的點狀出血及多發性血栓。本病的致病機制主要與病毒對血管內皮細胞的傷害導致的血管炎有關。根據研究,由常在性及誘導性一氧化氮合成酶所製造的一氧化氮具有許多重要的生理功能包括做為血管壁張力的釋放因子、神經傳導物質及宿主對病原的防禦因子。尤其對病毒的感染具有保護及細胞毒殺兩種雙極效應。由感染細胞產生的一氧化氮可以抑制痘病毒及反轉錄病毒的複製。然而當細胞中一氧化氮合成酶因狂犬病毒及里奧病毒感染大量表現時,高濃度的一氧化氮會引起嚴重發炎而傷害組織。因此我們提出調查猪瘟病毒對血管內皮細胞一氧化氮濃度的影響。本計畫分為兩年期,在生物體外進行研究。在不同時間及劑量下將猪瘟病毒感染猪主動脈內皮細胞。首先利用共軛焦顯微鏡來觀察細胞中常在性及誘導性一氧化氮合成酶的表現程度及分布形式。同時並使用PCR及西方點墨法來定量常在性及誘導性一氧化氮合成酶的表達及代謝速率。細胞所分泌出的一氧化氮量和常在性及誘導性一氧化氮合成酶的起動子活性將被分析。更進一步,常在性一氧化氮合成酶的磷酸化狀態將被分析。利用選擇性抑制劑來觀察PI3-Akt信號路徑、Src蛋白家族和細胞內鈣離子在常在性一氧化氮合成酶的磷酸化所扮演的角色。MAPK信號路徑在iNOS表現上的影響也將被探討。基於我們過去在研究的經驗,本計畫預期將被順利執行。其成果將幫助我們對猪瘟病毒的分子致病機轉的了解。同時也提供未來防疫上的一個新方向。
Classic swine fever (CSF) is a contagious disease of domestic pigs with high severity and mortality. It still causes a significant loss to pig industry in Taiwan even an extensive vaccination plan is implemented. Classic swine fever virus (CSFV) is a member of Pestivirus genus of Flaviridae. In acute and subacute cases, severe haemorrhagic diathesis and petechiation in multiple tissues and organs are derived from exaggerated inflammatory and immune responses and increased procoagulant activities. Vasculitis triggered by damages of endothelial cells when CSFV replicates inside constitutes the pathogenic basis of CSF. Nitric oxide (NO) identified as a vessel relaxing factor, a neurotransmitter, and a host defense mediator is generated from both a constitutive NO synthase (cNOS) and an inducible NO synthase (iNOS). Studies have indicated NO plays a role in both protective and cytotoxic responses in viral infections. NO produced by infected cells inhibited the replication of vaccinia virus and retroviruses. The highoutput NO from the up-regulated NO synthases followed cells infected with rabies virus and reovirus caused severe inflammations leading to tissue damages. A comprehensive study using culture cells for two years is proposed herein. Porcine aortic endothelial cells (PAEC) are infected with CSFV at different dosage and time dependent manners. The expression profiles and spatial localizations of cNOS and iNOS are checked by high resolution confocal laser scanning microscope. The expression levels and degradation rates of cNOS and iNOS are determined by semi-quantitative RT-PCR and Western blotting. The amount of nitric oxide and promoter activities of cNOS and iNOS are measured. Furthermore, the phosphorylation status of cNOS is examined. Selective inhibitors are used to determine the roles of PI 3-K/Akt pathway, Src protein, and intracellular calcium ions in eNOS phosphorylation. The expression of iNOS through MAPK pathway is analyzed. With past experiences in studies, this plan is surely to be conducted properly. This proposal will improve our understanding about molecular pathogenesis of CSFV and potentially contributes to develop a novel strategy for future prevention and therapy.
其他識別: NSC97-2313-B005-047-MY2
Appears in Collections:獸醫學系所



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