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研究生:張芝瑋
研究生(外文):Chih-wei Chang
論文名稱:神經壞死病毒誘發石斑魚細胞氧化壓力與病毒蛋白B1影響氧化壓力之探討
論文名稱(外文):Studies on betanodavirus induces oxidative stress and this stress modulated by viral protein B1 in GF-1 cells
指導教授:洪健睿洪健睿引用關係
指導教授(外文):Jiann-Ruey Hong
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物科技研究所碩博士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:98
中文關鍵詞:病毒蛋白B1抗氧化藥劑神經壞死病毒魚類細胞粒線體氧化壓力
外文關鍵詞:Betanodavirusantioxidantnonstructural protein B1reactive oxygen species (ROS)oxidative stress response
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神經壞死病毒(Nervous Necrosis Virus, NNV)屬於野田病毒(Betanodavirus)屬感染多種高經濟性魚種如石斑魚稚魚,並造成高死亡率。先前本實驗室研究發現紅點石斑神經壞死病毒RGNNV感染石斑魚細胞株造成粒腺體膜電位消失現象而使細胞壞死,是否病毒感染誘發粒腺體ROS產生而使粒線體失去其功能導致細胞的傷害,目前仍不清楚。因此,本論文將探討RGNNV引起氧化壓力之研究及病毒蛋白B1與氧化壓力之關係。
首先, RGNNV感染石斑魚細胞(GF-1)後觀察ROS變化,不論利用carboxy-H2DCFDA染色觀察細胞內ROS量,或Amplex Red偵測過氧化氫含量,結果顯示感染24小時有大量的ROS產生,並由粒線體染色追蹤ROS分布位置,ROS與粒線體位置有部分重疊。同時,RGNNV感染誘發宿主抗氧化機制啟動,隨感染時間增加而增加銅鋅型超氧歧化酶的蛋白表現。若添加抑制粒腺體ROS生成劑DPI (diphenyliodonium)或是 ROS清除劑NAC (N-acetylcysteine),能夠降低ROS量並且能穩定粒線體膜電位而提升細胞存活。
接著,我們希望藉由增加過氧化氫之代謝來降低細胞內氧化壓力,因此篩選出大量表現(overexpression)帶有斑馬魚之過氧化氫酶細胞株,並感染觀察,結果顯示感染後增加過氧化氫清除而能提升細胞的存活率。綜合以上結果,證實神經壞死病毒感染宿主細胞後經由ROS產生而造成粒線體氧化壓力,使粒線體膜電位失衡而導致細胞壞死。
除此之外,先前研究指出病毒蛋白B1具有抗細胞壞死之功能,由於 B1角色仍不清楚,為了釐清是否病毒蛋白B1參與氧化壓力之調節而減緩細胞死亡。因此,利用表現反義B1 RNA阻斷(knockdown) B1蛋白表現觀察其對氧化壓力之影響,結果顯示感染表現反義B1 RNA細胞能促進ROS的產生,並且銅鋅型超氧歧化酶及過氧化氫酶的抗氧酵素表現下降,說明B1蛋白與ROS之產生與調節抗氧化酵素有關。最後,利用北方墨點法發現B1能夠影響病毒的複製,大量表現(ovexpression)B1蛋白的石斑魚細胞株在感染早期12及24小時能抑制在病毒感染的RNA表現,反之,表現反義B1 RNA的細胞株感染後,則是可促進病毒RNA的表現。而B1是否經由影響病毒基因表現而影響氧化壓力,仍需更多證據釐清,希望本篇論文有助於神經壞死病毒造成氧化壓力研究,並應用於疾病控制上。
Betanodavirus is the causative agent of a highly infectious disease in larva of grouper fish and in other economic fishes and results in severe mortality and significant economic losses. In previous studies, RGNNV can induce necrotic cell death via mitochondrial membrane permeability (MMP) loss in fish cells. In this present, we want to know whether MMP loss connects to produce the reactive oxygen species (ROS) within oxidative stress.
First, to investigate that RGNNV could induce intracellular ROS production in grouper cells (GF-1), we used the fluorescent probe carboxy-H2DCFDA staining assay and Amplex Red to measure concentration of hydrogen peroxide. Our data shows that RGNNV infection leads to accumulation of the ROS at 24 p.i. RGNNV infection also induce antioxidant enzyme response, such as Cu/Zn SOD and catalase in GF-1. Further, either treatment with DPI (diphenyliodonuim), an inhibitor of NADPH oxidase activity, or NAC (N-acetylcysteine), a ROS scavenger, also can effectively block ROS production and reduce MMP loss in early-middle stages of replication cycle.
Second, we want to remove hydrogen peroxide by overexpression of catalase for reducing cell damage-induced by oxidative stress. Our data shows that overexpression of zebrafish catalase in GF-1 can prevent RGNNV-induced ROS production and delay cell death. These data suggest that RGNNV induces oxidative stress and mitochondria-mediated cell death may via ROS for triggering MMP loss in grouper cells.
On the other hand, the non-structure proteins B1 which play a role on anti-necrotic death function. To understand how protein B1 effect between cell death and oxidative stress are examined. In the present, knockdown of B1 expression by antisense RNA promoted ROS production and decreased Cu/Zn SOD and catalase expression. Moreover, we found that protein B1 can modulate viral replication by northern blot analysis, which reduce viral replication at 12 and 24 p.i. Our results suggest that RGNNV can induce the oxidative stress response and viral protein B1 can against oxidative injury. These results provide insights into RGNNV infection pathogenesis that provide a firm theoretical basis for the investigation of antioxidant therapies in this important disease in future.
目錄
中文摘要..................................................................................................... I

英文摘要..................................................................................................... III

誌謝............................................................................................................. V

目錄............................................................................................................. VI

表圖目錄..................................................................................................... IX

縮寫表......................................................................................................... XI

第一章、 緒論
第一節、台灣石斑魚養殖概況.................................................................. 1
第二節、神經壞死病毒背景說明............................................................... 2
第三節、細胞壞死與細胞凋亡................................................................... 4
第四節、病毒感染與細胞凋亡之研究....................................................... 5
第五節、氧化壓力與細胞死亡................................................................... 8
第六節、病毒與氧化壓力的關係............................................................... 10
第七節、研究之目的................................................................................... 12

第二章、材料與方法
第一節、實驗材料....................................................................................... 13
第二節、實驗儀器....................................................................................... 22
第三節、研究方法及實驗步驟................................................................... 24
第三章、結果
第一節 神經壞死病毒感染石斑魚背鰭細胞造成ROS產生................... 38
第二節 神經壞死病毒感染後誘導宿主抗氧化酵素Cu/ Zn SOD和catalase蛋白之表現......................................................................
39
第三節 處理抗氧化藥劑對神經壞死病毒感染所造成的氧化壓力之影響....................................................................................................
40
第四節 處理抗氧化藥劑對神經壞死病毒感染所造成的細胞壞死之影響....................................................................................................
41
第五節 表現斑馬魚過氧化氫酶catalase細胞株對細胞壞死的影響...... 44
第六節 抗壞死病毒蛋白B1對氧化壓力的影響...................................... 45
第七節 抗壞死病毒蛋白B1對病毒複製的調控...................................... 47

第四章、討論
第一節 神經壞死病毒感染促進ROS產生扮演的功能.......................... 49
第二節 神經壞死病毒感染對抗氧化酵素的調節................................... 50
第三節 抗氧化藥劑或是大量表現catalase對細胞壞死之影響............. 51
第四節 非結構病毒蛋白B1對氧化壓力之調控角色.............................. 51
第五章、未來展望...................................................................................... 53
參考文獻..................................................................................................... 54
圖表附錄..................................................................................................... 67
自述............................................................................................................. 98
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