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系統識別號 U0026-0408201410493300
論文名稱(中文) 神經壞死病毒所誘導之活性氧分子對於自噬作用的調節
論文名稱(英文) The effects of RGNNV-induced reactive oxygen species production on autophagic processing regulation
校院名稱 成功大學
系所名稱(中) 生物科技研究所
系所名稱(英) Institute of Biotechnology
學年度 102
學期 2
出版年 103
研究生(中文) 陳博榕
研究生(英文) Po-Jung Chen
學號 L66014067
學位類別 碩士
語文別 中文
論文頁數 96頁
口試委員 指導教授-洪健睿
口試委員-耿全福
召集委員-黃玲惠
口試委員-劉校生
中文關鍵字 神經壞死病毒  活性氧分子  自噬作用  抗氧化藥劑  mTOR  病毒複製 
英文關鍵字 Betanodavirus (RGNNV)  reactive oxygen species  autophagy  antioxidants  mammalian target of rapamycin (mTOR)  viral replication 
學科別分類
中文摘要 自噬作用為細胞內維持恆定的代謝機制,可將老舊或受損胞器分解回收,以提供能量合成新的胞器或其他用途。而當細胞面臨壓力或病原入侵時也會誘導發生。實驗室先前研究證實神經壞死病毒(RGNNV)感染石斑魚鰭細胞株(GF-1)會產生由活性氧分子(O2-, H2O2)所介導的自噬作用。因此本研究想要探討在病毒感染下所誘導之活性氧分子如何調節自噬作用。首先由蛋白質表現分析結果顯示,病毒感染使自噬作用上游分子mTOR活性上升,並由AMPK及Akt所調控。若添加抗氧化劑DPI(Diphenylene iodonium)抑制O2-生成能減少mTOR活性,而添加GSH(Glutathione)促H2O2代謝則無法降低mTOR活性。接著進一步由穩定表現mRFP-LC3細胞株及MDC染色觀察病毒感染所誘導之自噬作用,結果發現,抑制O2-生成或促進H2O2代謝均能降低自噬作用,其中又以抑制O2-能降低的效果最為明顯,且由蛋白質表現分析也觀察到抑制O2-能降低Class III PI3K(Vps34)的表現量。在病毒效價及細胞存活方面,抑制O2-能降低病毒效價並提高細胞存活。 此外抗氧化轉錄因子Nrf2,近來文獻顯示會和自噬作用中的sequestosome 1/p62 (SQSTM1/p62)蛋白具有交互作用以調節細胞中氧化壓力。在我們系統中亦發現,若降低病毒感染所誘導的活性氧分子,Nrf2及p62的表現會減少。若進一步利用Nrf 2抑制劑RA(Retinoic Acid)或p62 siRNA,顯示 Nrf2和p62在RGNNV感染下有交互作用。總結以上結果可知,O2-為病毒感染所誘導的主要活性氧分子,並藉由調節mTOR活性及Class III PI3K(Vps34)之表現,參與病毒誘發的自噬作用及細胞死亡,另外也調節Nrf2及抗氧化酵素的表現。
英文摘要 Autophagy is a cellular metabolic process to degrade and recycle the older or damaged organelle that autophagic also induces under the stress or virus infection. In this study, we found that (1) antioxidant DPI (for blocking superoxide production) treatment with RGNNV-infected GF-1 cells can block mTOR activity, but did not blocked by GSH (for reducing hydrogen oxide production) treatment; (2) with MDC staining in mRFP-LC3 cells, we found that antioxidant DPI treatment can reduce O2- production and that can significantly reduce virus-induced autophagy and enhance cell viability; (3) we found that Class III PI3K(Vps34) also participates in virus-induced autophagy by reactive oxygen species. Finally, we have found that antioxidant transcription factor Nrf2 can interact with p62 by using specific inhibitor retinoic acid or p62 siRNA treatment in GF-1 cells with RGNNV infection. Taken together our results suggest that RGNNV-induced ROS (O2-) signals can regulate the autophagic process for enhancing viral replication and host cell death. Then, the autophagic process is regulated by mTOR and class III PI3K signals. Then, the mTOR signal is regulated by either AKT, or AMPK activities. Our finding provided insight into RNA virus molecular mechanism.
論文目次 摘要 I
Abstract II
誌謝 VII
圖表目錄 XIII
附錄目錄 XV
縮寫表 XVI
第一章、緒論 1
第一節、台灣石斑魚養殖概況 1
(一) 石斑魚背景簡介 1
(二) 台灣的石斑魚養殖現況與瓶頸 2
第二節、神經壞死病毒背景簡介 2
(一) 神經壞死病毒之發現 3
(二) 神經壞死病毒之命名與分類 3
(三) 感染神經壞死病毒之病理特徵 4
(四) 神經壞死病毒之基因與蛋白質功能之研究 4
(五) 神經壞死病毒與細胞死亡之研究 5
第三節、活性氧分子(ROS) 7
(一) 活性氧分子的產生與來源 7
(二) 活性氧分子與氧化壓力(Oxidative stress) 8
(三) 活性氧分子與自噬作用之調控 8
第四節、自噬作用 9
(一) 自噬作用的發現 9
(二) 自噬作用的型式 10
(三) 自噬作用的機制與調控 10
第五節、自噬作用與mTOR蛋白 11
(一) mTOR蛋白與其抑制劑Rapamycin的發現 12
(二) mTOR蛋白的訊號傳遞與細胞內功能 12
第六節、自噬作用與病毒感染之互動 14
(一) 病毒引起之自噬作用 14
(二) 自噬作用影響病毒增值 14
第七節、研究目的與動機 16
第二章、材料與方法 17
第一節、實驗材料 17
(一) 生物材料 17
(二) 抗體 17
(三) 細胞用試藥 18
(四) 生物反應試劑組(Kits) 19
(五) 載體 20
(六) p62 primer 20
(七) p62 siRNA序列 20
(八) 細菌培養基與抗生素之配製 21
(九) 細胞培養液配製 21
(十) 蛋白質樣本萃取溶液 22
(十一) 蛋白質電泳及膠體製作緩衝溶液 23
第二節、實驗儀器 26
第三節、實驗方法及步驟 28
(一) 菌種的培養及儲存 28
(二) 質體DNA的萃取 28
(三) 聚合酵素連鎖反應 (PCR) 29
(四) 膠體萃取 30
(五) 質體剪接及黏合反應 30
(六) 轉形反應(Transformation) 31
(七) 細胞培養 32
(八) 病毒增值與效價測定 34
(九) 蛋白質表現量測定 35
(十) MDC染色偵測自噬溶酶體 37
第三章、結果 38
第一節、RGNNV感染GF-1對mTOR活性的影響 38
第二節、病毒感染所誘導之氧化壓力對mTOR活性的調節 39
第三節、病毒感染所誘導之氧化壓力對自噬作用的調節 40
第四節、RGNNV感染所誘導之氧化壓力對細胞死亡的影響 41
第五節、自噬作用的完整性對於病毒複製的影響 42
(一) 抑制p62能減少病毒誘導之細胞死亡 42
(二) 抑制溶酶體與自噬體的融合能減少病毒蛋白表現 43
第六節、RGNNV所誘導之氧化壓力與抗氧化系統的調節 43
(一) Nrf2參與病毒感染之抗氧化壓力調節 44
(二) Nrf2與p62/SQSTM1間的抗氧化壓力調節 44
第四章、討論 46
第一節、活性氧分子對自噬作用的調節 46
第二節、神經壞死病毒所誘發之自噬作用 47
第三節、自噬作用對病毒感染的多元角色 48
第五章、未來展望 50
參考文獻 51
圖表 60
附錄 89
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