臺灣博碩士論文加值系統

腸病毒 71 型是屬於小的 RNA 病毒的一種，臨床上一般輕微的症狀有手足口症及疱疹性咽唊炎，嚴重導致腦神經系統病變，故致病及致死率都相當高，目前為止並沒有一個有效的抗病毒藥物及疫苗來預防。先前研究指出腸病毒 71 型與氧化壓力有相關，當病毒感染時宿主細胞會產生大量的活性氧物質，本研究認為傳統中國藥方對於宿主本身具有一些治療的靶點，許多文獻證實荊芥具有抗氧化及抗發炎之功效，本研究證實了荊芥對於腸病毒 71 型感染具有保護能力，擬進一步使用代謝體學方法去探討其保護機制。由代謝體學結果發現腸病毒 71 型感染後穀胺醯胺明顯增加，先特別針對穀胺醯胺的作用機制去研究，結果發現當穀胺醯胺含量較低時，病毒感染能力、細胞病變情形及病毒複製能力皆明顯下降，同時也發現腸病毒71型感染能活化穀胺醯胺代謝路徑中的轉氨酶、谷氨酸脫氫酶、胺基甲醯磷酸鹽合成酶，故結果證實穀胺醯胺代謝路徑對於腸病毒71型感染扮演重要角色之一。最後利用代謝體學方式去探討荊芥對於腸病毒71型保護機制，結果發現荊芥保護效果主要影響的代謝路徑為穀胱甘肽代謝路徑、穀胺醯胺代謝路徑及半胱氨酸和甲硫氨酸代謝路徑，感染腸病毒71型後其穀胱甘肽與氧化穀胱甘肽比值明顯下降、ATP 生成量也下降及同型半胱胺酸含量上升，以上結果即證明經腸病毒感染後氧化壓力上升，加入荊芥後反之，證實荊芥對於腸病毒71型保護機制與氧化壓力代謝路徑息息相關。

Enterovirus 71 (EV71) is a non-enveloped RNA virus within the family Picornaviridae and is one of the causative viral agents of hand, foot, and mouth disease (HFMD). Infection with EV71 may also cause severe neurological diseases, such as acute encephalitis, acute flaccid paralysis, and even cause death. However, there is no effective vaccine or antiviral treatment available thus far. Previous studies demonstrated that oxidative stress enhances viral replication and cytopathic effects (CPE) in infected cells, which leads to increased production of reactive oxygen species (ROS). Schizonepeta tenuifolia Briq. (STE) is a Chinese herbal decoction that is used as a cure for the common cold, fever, headache, and poor circulation. STE have been known to display immunomodulatory and antioxidant effects. Our study has shown that antiviral effects of STE on Enterovirus 71. Therefore, we have employed the metabolomic approach to investigate the antiviral effects of STE on Enterovirus 71. After bioinformatic and statistical analyses, we mapped the metabolites, the levels of which changed during infection, onto the biochemical pathways. The results showed that the STE primarily effects through modulation of glutathione metabolic pathway, glutamine and cysteine and methionine metabolic pathways. These findings suggest that STE may suppress viral replication via modulation of cellular redox milieu.

指導教授推薦書…………………………………………………………
口試委員會審定書…………………………………………………………
長庚大學博碩士論文著作授權書…………………………………………………………iii
誌謝…………………………………………………………iv
中文摘要…………………………………………………………v
英文摘要…………………………………………………………vii
目錄…………………………………………………………viii
圖目錄…………………………………………………………xii
第一章 序論
1.1 研究背景
1.1.1 腸病毒71型…………………………………………………………1-4
1.1.2 荊芥…………………………………………………………4-5
1.1.3 代謝體學…………………………………………………………5-7
1.1.4 穀胺醯胺…………………………………………………………7-9
第二章 材料與方法
2.1 藥品、緩衝液、培養液及試劑…………………………………………………………10
2.1.1細胞及病毒…………………………………………………………10
2.1.2中藥…………………………………………………………10
2.1.3 細胞培養…………………………………………………………10-11
2.1.4 細胞計數…………………………………………………………11
2.1.5 代謝體學測試…………………………………………………………11
2.1.6 細胞存活率測試…………………………………………………………12
2.1.7 病毒斑測試…………………………………………………………12
2.1.8 蛋白質萃取定量與西方點墨法…………………………………………………………13-15
2.1.9 RNA萃取、反轉錄聚合酶鏈反應、定量聚合酶鏈反應…………………………………………………………15
2.1.10 小分子干擾核糖核酸…………………………………………………………15
2.1.11 細胞病變染色…………………………………………………………16
2.2 實驗方法…………………………………………………………16
2.2.1 細胞培養…………………………………………………………16
2.2.2 病毒液收集…………………………………………………………17
2.2.3 代謝體學測試…………………………………………………………17-18
2.2.4 小分子干擾核糖核酸…………………………………………………………18-19
2.2.5 細胞存活率測試…………………………………………………………19
2.2.6 病毒斑測試…………………………………………………………19-20
2.2.7 定量聚合酶鏈反應…………………………………………………………20-21
2.2.8 蛋白質萃取定量與西方點墨法…………………………………………………………21-22
2.2.9 細胞核染色…………………………………………………………22
2.2.10 荊芥配製…………………………………………………………22
第三章 結果…………………………………………………………23
3.1 腸病毒 71 型感染宿主細胞代謝體學分析…………………………………………………………23
3.2 穀胺醯胺促進腸病毒 71 型感染細胞之死亡與細胞病變效應…………………………………………………………24-25
3.3 穀胺醯胺增強腸病毒 71 型複製…………………………………………………………25
3.4 確認穀胺醯胺影響宿主細胞經腸病毒 71 型複製…………………………………………………………25-26
3.5 宿主細胞經由腸病毒71型感染後可增加穀胺醯胺酶、穀氨酸鹽脫氫脢及胺基甲醯磷酸鹽合成酶…………………………………………………………26-27
3.6 經小分子干擾核糖核酸默化穀胺醯胺酶、穀氨酸鹽脫氫脢及胺基甲醯磷酸鹽合成酶之宿主細胞，較不容易受腸病毒71 型感染…………………………………………………………27-28
3.7 經小分子干擾核糖核酸默化穀胺醯胺酶、穀氨酸鹽脫氫脢及胺基甲醯磷酸鹽合成酶處理之宿主細胞在腸病毒71型感染後的細胞病變程度…………………………………………………………28
3.8 經小分子干擾核糖核酸默化穀胺醯胺酶、穀氨酸鹽脫氫脢及胺基甲醯磷酸鹽合成酶之宿主細胞中，病毒複製程度降低…………………………………………………………29
3.9 荊芥有效抑制腸病毒71型複製能力…………………………………………………………29
3.10 荊芥處理後對於腸病毒 71 型感染宿主細胞代謝體學分析…………………………………………………………30
3.11 荊芥抗病毒主要影響之代謝路徑…………………………………………………………30-31
第四章 討論…………………………………………………………32-39
參考文獻…………………………………………………………40-50
圖…………………………………………………………51-64
表格…………………………………………………………65-68

圖 目錄
圖一、腸病毒 71 型感染宿主細胞代謝體學分析
圖二、穀胺醯胺促進腸病毒 71 型感染細胞之死亡與細胞病變效應
圖三、穀胺醯胺增強腸病毒 71 型複製
圖四、穀胺醯胺濃度對宿主細胞經腸病毒 71 型複製的影響
圖五、宿主細胞經由腸病毒 71 型感染後可增加穀胺醯胺酶 (GLS)、穀胺酸鹽脫氫脢 (GDH) 及胺基甲醯磷酸鹽合成酶 (CAD2)
圖六、經小分子干擾核糖核酸默化穀胺醯胺酶 (GLS)、穀胺酸鹽脫氫脢(GDH)及胺基甲醯磷酸鹽合成酶 (CAD2) 之宿主細胞，較不容易受腸病毒 71 型感染
圖七、經小分子干擾核糖核酸默化穀胺醯胺酶 (GLS)、穀氨酸鹽脫氫脢(GDH)及胺基甲醯磷酸鹽合成酶 (CAD2) 處理之宿主細胞在腸病毒 71 型感染後的細胞病變程度
圖八、經小分子干擾核糖核酸默化穀胺醯胺酶 (GLS)、穀氨酸鹽脫氫脢(GDH)及胺基甲醯磷酸鹽合成酶 (CAD2) 之宿主細胞中，病毒複製程度降低
圖九、荊芥有效抑制腸病毒 71 型複製能力
圖十、荊芥處理腸病毒 71 型感染之宿主細胞的代謝體學分析
圖十一、荊芥抗腸病毒 71 型主要影響之代謝路徑及變化量

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