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研究生：	陳顗同 Chen, I-Tung
論文名稱：	寄主細胞粒線體與白點症病毒致病機轉之相關性 Virus-host interaction: the role of mitochondria in the pathogenesis of white spot syndrome virus infection
指導教授：	王涵青 Wang, Han-Ching
學位類別：	碩士 Master
系所名稱：	生物科學與科技學院 - 生物科技研究所 Institute of Biotechnology
論文出版年：	2010
畢業學年度：	98
語文別：	中文
論文頁數：	81
中文關鍵詞：	白蝦、白點症病毒、粒線體、代謝質體學
外文關鍵詞：	Litopenaeus vannamei, WSSV, Mitochondria, Metabolomics
相關次數：	點閱：154 下載：1
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台灣過去素有「草蝦王國」的美名，但於1990年代所爆發的蝦白點症徹底瓦解了台灣的草蝦產業，直到今日科學家們對於解決白點症病毒感染問題依舊束手無策。根據研究蝦白點症病毒感染後白蝦胃組織圖譜分析(protein profile)以及白點症病毒感染後蝦類淋巴組織基因表現微陣列分析(microarray)之結果，推測蝦類粒線體在白點症病毒感染後應扮演重要角色。粒線體相關的蛋白質中，粒線體外膜主要蛋白-電位調控型離子通道(voltage-dependent anion channel, VDAC)於白點症病毒感染後表現量明顯上升，而其上游調控基因hexokinase表現量呈現下降趨勢。這種白點症病毒感染後引發 hexokinase 與 VDAC 之間失衡狀態，可能造成粒線體通透性孔洞過度開啟，導致粒線體膜電位喪失、內外離子平衡破壞、 ATP/ADP 能量代謝異常、粒線體胞器腫脹及破裂、產生大量活性氧物質(ROS)，驅使細胞死亡。本研究目的在於建構白點症病毒於粒腺體層次上的致病機轉以及宿主對於病毒感染後之反應相關研究。實驗結果發現白點症病毒感染時程以24小時為一複製循環，蝦類於白點病毒感染後12至24小時間，病毒進行快速複製，醣類代謝路徑受到改變，快速行使類似 Warburg effect 之醣類乳酸發酵反應，造成醣解中間反應物累積，促進代謝途徑轉至五碳醣循環反應，提供病毒複製所需物質，此外細胞脂質代謝被大量促進，推測提供細胞替代性之能量來源。於病毒第一個複製循環末期，即病毒將進行釋出動作之時，此時醣類代謝活動被抑制、粒線體膜電位喪失、 ADP/ATP 比值高等現象，將細胞趨於功能化喪失導致其走向死亡。本研究所得為白點症病毒感染過程中，建立新穎性方向之致病機轉，期望未來可藉由更深入的研究，釐清相關細胞代謝路徑於白點症病毒感染後之改變，從中思考對於疾病的治療及防範策略。

White spot syndrome virus (WSSV) is the causative pathogen of white spot disease (WSD), a disease with great impact on the cultured shrimp industry. Pathogenesis of WSSV, however, is poorly understood. It has been postulated that mitochondria may play an important role during WSSV pathogenesis, and that mitochondrial dysfunction may be involved in cell death. Materials move in and out of the mitochondria via the mitochondrial permeability transition pore (MPTP). The outermost area of the pore is critically important in permeability and is composed of a single protein, the voltage dependent anion channel (VDAC) protein. Previous studies found that host VDAC is upregulated after WSSV infection, suggesting a mitochondrial role in pathogenesis. Preliminary microarray analysis showed that hexokinase was down-regulated after WSSV infection. Hexokinase mediates the opening of MPTP, and is also involved in a key metabolic pathway, the glycolytic pathway. There are 3 parts to this study. Because imbalanced VDAC-hexokinase interaction may result in mitochondrial membrane permeabilization (MMP), the study is the first investigation about the impact of WSSV on mitochondrial activity. Three factors (mitochondrial membrane potential, energy production, and oxidative stress) were assessed over a pre- and post-infection time period (0, 12, 24, 36, 48, 60 and 72 hours post-infection). The data showed that beginning at 24h post-WSSV challenge, and increasing over time, host hemocytes showed loss of mitochondrial membrane potential. Also beginning 24 h post-infection, energy production was disrupted (shown in increased ADP/ATP ratio). No oxidative stress was detected. This clearly shows that mitochondrial function was disrupted. Because hexokinase is also involved in glycolysis, fatty acid metabolism and the pentose phosphate pathway (PPP), the second part of this study analyzed their post-infection changes. Amounts of glucose and lactate (for glycolysis), triglycerides (for fatty acid metabolism) and the activity of G6PDH, the key enzyme in the pentose phosphate pathway (PPP) were measured at the same post-infection time points. The data show that at 24 h post-infection glucose concentration was higher than in control group. This indicates that glycolysis was down-regulated. Beginning at 12 h, triglyceride concentration in the infected group was significantly lower than in the control group. G6PDH activity was higher than the control group at 12 and 36 h. As the infection progressed, there was a temporal flux in metabolites. In order to clarify the viral life cycle, and explain the metabolic flux, we further quantified WSSV replication efficiency and found that replication efficiency was highest at 24 h. It appears that the replication cycle and the changed metabolic flux are parallel. Overall the results of this study support the view that viral replication and release are clearly related to metabolic changes in the cell as well as changes in mitochondrial activity.

中文摘要	3
Abstract	5
誌謝	8
目錄	10
前言	12
材料與方法	21
白點症病毒來源、感染試驗及實驗動物	21
白點症病毒致死濃度(lethal dose)測試	21
Hexokinase 基因於白蝦 L. vannamei 體內表現之組織特異性(tissue tropism)	22
利用蝦體內基因沉默化探討目標致病機轉路徑在病毒感染過程之相關性	24
觀測蝦血球細胞之粒線膜電位在白點症病毒感染過程的變化	25
觀測蝦血球細胞之 ADP/ATP 比值在白點症病毒感染過程的變化	27
觀測蝦血球細胞之 ROS 在白點症病毒感染過程的變化	28
觀測蝦血球細胞之 H2O2 濃度在白點症病毒感染過程的變化	28
蝦血清中 glucose 濃度於白點症病毒感染過程的變化	29
蝦血清中 lactate 濃度於白點症病毒感染過程的變化	30
蝦血漿中 triglyceride 濃度於白點症病毒感染過程的變化	31
觀測蝦血球細胞之 G6PDH 酵素活性在白點症病毒感染過程的變化	32
觀察蝦類組織內於白點症染病毒過程之病毒顆粒累積變化	33
結果	36
白點症病毒致死濃度(lethal dose)測試結果	36
Hexokinase基因普遍表現於於白蝦體內之各組織	36
蝦體內hexokinase基因沉默化不會促進或是降低白點症病毒感染所導致之死亡率	37
蝦血球細胞初代培養平台之建立	38
白點症病毒感染過程造成蝦血球細胞之粒線膜電位喪失	39
白點症病毒感染造成蝦血球細胞具有較高的ADP/ATP比值	40
白點症病毒感染並不會造成蝦血球細胞 ROS 的累積	41
白點症病毒感染並不會造成蝦血球細胞 H2O2 的累積	41
蝦血清中 glucose 濃度於白點症病毒感染過程末期大量下降	42
蝦血清中 lactate 濃度於白點症病毒感染過程末期大量下降	43
蝦血清中 triglyceride 濃度於白點症病毒感染過程中皆大量下降	44
在白點症病毒感染過程之特定時間點可偵測到蝦血球細胞中之 G6PDH酵素具有較高活性表現	45
白點症病毒於感染後蝦類組織內之累積情況	46
討論	48
參考文獻	60
實驗圖表	65
Table 1、 PCR primers used in this study	65
Figure 1、白點症病毒感染源致死濃度劑量之建立	66
Figure 2、蝦體內 hexokinase 基因表現量之組織特異性比較	67
Figure 3、蝦體內 hexokinase 基因沉默化不會促進或是降低白點症病毒感染所導致之死亡率	68
Figure 4、初代培養之蝦血球細胞於顯微鏡視野下之三種蝦血球細胞型及細胞形態	69
Figure 5、白點症病毒感染過程造成蝦血球細胞之粒線膜電位喪失	70
Figure 6、白點症病毒感染後蝦血球細胞之 ADP/ATP 比值	71
Figure 7、白點症病毒感染不會造成蝦血球細胞 ROS 的累積	72
Figure 8、白點症病毒感染不會造成蝦血球細胞 H2O2 的累積	73
Figure 9、白點症病毒感染過程中蝦血清 glucose 濃度之變化	74
Figure 10、白點症病毒感染過程對蝦血漿中 lactate 之變化	75
Figure 11、白點症病毒感染過程中蝦血漿 triglyceride 濃度之變化	76
Figure 12、白點症病毒感染過程中，蝦血球細胞 G6PDH 酵素活性表現之變化	77
Figure 13、蝦類組織內於白點症染病毒過程之病毒顆粒累積變化	78
Figure 14、本研究中所建立之白點症病毒代謝路徑改變假說圖	79
附錄1. 白點症病毒在 mitochondria transition pore 的調節假說圖	80
附錄2. Mitochondria transition pore過度開啟後所導致之數個mitochondria-mediate cell death 路徑圖	81

                                    

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校內：2020-12-31公開
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