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研究生：	黃韻慈 Huang, Yun-Tzu
論文名稱：	由白點症病毒所誘發之瓦氏效應分析其主要致病機制 WSSV pathogenesis: Roles of the induced Warburg effect
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：	2012
畢業學年度：	100
語文別：	中文
論文頁數：	118
中文關鍵詞：	白蝦、白點症病毒、瓦氏效應、蛋白質體學、雷帕黴素標靶蛋白訊息傳遞路徑
外文關鍵詞：	Litopenaeus vannamei, White spot syndrome virus, Warburg effect, Proteome, mTOR signaling pathway
相關次數：	點閱：144 下載：0
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90年代之際，由於過高的養殖密度及缺乏適當的經營管理，導致蝦類病毒性疾病的爆發，使得全球養蝦產業面臨瓦解之危機。其中又以由白點症病毒 (WSSV)所造成的白點症 (WSD)為最嚴重的蝦類病毒性疾病，可於短時間內造成蝦類大量死亡。過去研究由WSSV感染前後的蝦體蛋白質差異性表現圖譜發現，宿主粒線體之代謝路徑於病毒感染後有轉變的趨勢；另在代謝體學分析中，指出蝦體於感染WSSV後，可觀察到葡萄醣大量被利用而乳醣大量累積的現象。綜合以上結果，我們推測於WSSV感染後，將誘導宿主代謝路徑走向類似癌細胞中發現之Warburg effect (或稱作有氧醣解作用)，因而本實驗希望由WSSV所誘發之Warburg effect分析其主要致病機制。本實驗首先利用即時定量PCR偵測可調控Warburg effect的重要因子，結果於WSSV感染過程皆呈現差異性表現。接續將粒線體相關蛋白voltage dependent anion channel (VDAC)進行基因默化後觀測其Warburg effect，但由於控制組之Warburg effect亦有減緩之現象，因此無法利用基因默化之實驗釐清VDAC於白點症病毒誘發之Warburg effect中所扮演之角色。本實驗接續利用具有高靈敏度之label-free蛋白質體學系統對於注射WSSV及生理食鹽水 (PBS)之蝦血球細胞蛋白質進行高通量分析，總共鑑定出約八百種蝦類蛋白質，其中超過六百個具有顯著差異性表現，並且參與能量代謝之酵素於病毒感染早期有上升之趨勢，皆符合 Warburg effect。另外，於本實驗蛋白體學研究亦發現，可活化 mTOR之蛋白質 Rheb其表現量於病毒感染早期及晚期有極大之差異，mTOR訊息傳遞路徑為推動整體 Warburg effect的發生及調控轉譯作用的重要因子。進一步利用 mTOR抑制劑處理發現病毒的基因表現及病毒顆粒累積皆受到影響，推測 mTOR訊息傳遞路徑可能於白點症病毒致病機制中扮演重要角色。本實驗驥望藉由釐清 WSSV之致病機轉，進而發展白點症之治療與防治之策略。

White spot disease (WSD) is a treat to the global shrimp industry that results in enormous economic losses worldwide. White spot syndrome virus (WSSV), the causative agent of WSD, is a novel, large dsDNA virus showing only a few similarities to current known viruses. Because of its uniqueness, the infection model of WSSV needs to be studied ab initio. In this study, we used systems biology to study the global interactions between WSSV and its host. Systems biology is an integrated approach that acquires and assembles high-throughput information into a database; develops and uses computational algorithms to analyze these data; and then designs experimental systems to test the hypothetical models. This approach allows us to construct a global cell pathway network that shed new light on the puzzle of virus-host interactions. To date, we have already known that that mitochondrial metabolism was very likely to be involved in the WSSV replication cycle. Moreover, by measuring several factors in different metabolic pathways, we found that WSSV induces distinct cellular responses at different replication stages; specifically, the Warburg effect in the WSSV genome replication stage, and cell death in the late stage.
In this research, we used a label-free quantitative proteomic approach to identify in vivo alterations in the proteome of shrimp hemocytes at different stages of WSSV infection. After integrating time series of proteomic profiles with computational modeling approaches to identify the key changes that WSSV infection, a global metabolic network of shrimp hemocyte cells was produced. In this network, several characteristics fits the Warburg effect, such as a shift from glycolysis to the pentose phosphate pathway. To understand the potential role of these alterations to WSSV pathogenesis, molecular and animal experiments were subsequently conducted. By using specific inhibitors and dsRNA mediated RNA interference to block the activity of the putative bottlenecked factors, we found that mTOR pathway play an important role for WSSV pathogenesis and WSSV-induced Warburg effect. We believe the work is very important both to science and to the aquaculture industry.

中文摘要	I
Abstract…	III
誌謝……	V
目錄……	VII
表目錄…	X
圖目錄….	XI
前言……	1
材料與方法	13
1.	偵測調控Warburg effect之重要因子於病毒感染過程之表現差異	13
1-1實驗病毒來源及實驗動物	13
1-2採樣流程	13
1-3 cDNA模板製備方式	14
1-4 即時定量PCR分析方式	14
2.	利用RNAi技術平台分析VDAC於WSSV所誘發之Warburg effect中所扮演之角色	15
2-1 dsRNA製備	15
2-2實驗動物及注射劑量	16
2-3 測量血清中之葡萄醣含量	17
2-4 測量血清中之乳醣含量	17
2-5 測量病毒顆粒累積變化於粒線體相關蛋白VDAC默化後感染白點症病毒期間之變化	18
3.	白點症病毒感染前後蛋白質圖譜之變化	19
3-1 白點症病毒感染之檢測	19
3-2 蝦血球細胞蛋白質萃取及定量	20
3-3 蝦血球細胞蛋白質之分析	22
3-4 蛋白質體學結果分析	23
4.	利用抑制劑解析mTOR訊息傳遞路徑對於WSSV致病機制之重要性	24
4-1 預先利用 mTOR抑制劑處理後再感染白點症病毒觀察磷酸化之4E-BP蛋白質表現量	24
4-2 白點症病毒感染後利用mTOR抑制劑rapamycin處理蝦隻觀察死亡率	26
4-3 預先利用mTOR抑制劑rapamycin處理後再感染白點症病毒觀察死亡率	26
4-4 預先利用mTOR抑制劑rapamycin處理後再感染白點症病毒觀察觀察病毒基因表現量	27
4-4 預先利用 mTOR抑制劑 rapamycin處理後再感染白點症病毒觀察觀察病毒顆粒累積量	28
4-5 預先利用 mTOR抑制劑 Torin1處理後再感染白點症病毒觀察病毒基因表現量	28
4-6 預先利用mTOR抑制劑Torin1處理後再感染白點症病毒觀察觀察病毒顆粒累積量	29
結果……	31
1. 調控Warburg effect之重要因子於病毒感染過程之表現差異	31
1-1 VDAC及Hexokinase基因於感染白點症病毒後之表現量	31
1-2 白蝦Pyruvate kinase基因三種isoform於感染白點症病毒後之表現量	31
1-3 調控醣解作用之重要因子Hypoxia induce factor-1α於病毒感染過程之表現差異	32
1-4 調控醣解作用之重要因子Thioredoxin於病毒感染過程之表現差異	33
2. VDAC於WSSV所誘發之Warburg effect中所扮演之角色	33
2-1 宿主genome中之累積病毒顆粒與病毒複製速率於粒線體相關蛋白VDAC默化後感染白點症病毒期間之變化	34
2-2 蝦血清中之葡萄醣及乳醣濃度於粒線體相關蛋白VDAC默化後於白點症病毒致病期間之變化	34
3. 白點症病毒感染前後蛋白質圖譜之變化	35
3-1 白點症病毒檢測結果	35
3-2 蝦血細胞蛋白質品質確認結果	36
3-3 感染白點症病毒之蝦血球細胞蛋白質分析	37
3-3-1	參與醣解作用與五碳醣磷酸化路徑之酵素於白點症病毒感染早期 (12 hpi)大量上升	38
3-3-2	部分參與檸檬酸循環之酵素於白點症病毒感染早期 (12 hpi)大量上升...	38
3-3-3	參與脂質代謝之酵素於白點症病毒感染早期大量上升	39
3-3-4	參與調控醣解作用及轉譯作用之訊息傳遞路徑之酵素於白點症病毒感染早期大量上升	39
3-3-5	活化mTOR信息傳導路徑相關之細胞蛋白質Rheb於病毒感染早期大量上升	40
4. mTOR訊息傳遞路徑對於WSSV致病機制之重要性	41
4-1預先利用 mTOR抑制劑處理後再感染白點症病毒之磷酸化4E-BP蛋白質表現量	41
4-2 白點症病毒感染後利用mTOR抑制劑rapamycin處理蝦隻之死亡率	42
4-3 預先利用mTOR抑制劑rapamycin處理後再感染白點症病毒之死亡率	42
4-4 rapamycin處理後再感染白點症病毒之蝦血清中乳醣濃度變化	43
4-5 rapamycin處理後再感染白點症病毒之病毒基因表現量	43
4-6 rapamycin處理後再感染白點症病毒之病毒顆粒累積現象	44
4-7 Torin1處理後再感染白點症病毒之病毒基因表現量	44
4-8 Torin1處理後再感染白點症病毒之病毒顆粒累積現象	46
討論........	47
參考文獻	59
圖表........	65
附錄........	111

                                    

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校內：2022-12-31公開
校外：不公開電子論文尚未授權公開，紙本請查館藏目錄

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