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研究生:張以暄
研究生(外文):Yi-Hsuan Chang
論文名稱:研究腸病毒71型2BC蛋白引發細胞自噬的機制
論文名稱(外文):Mechanistic study on Autophagy Induced by Expressions of the Enterovirus 71 2BC Protein
指導教授:龔思豪
指導教授(外文):Szu-Hao Kung
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術暨檢驗學系
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:68
中文關鍵詞:腸病毒複製鈣離子細胞自噬
外文關鍵詞:Enterovirus replicationcalciumAutophagy
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細胞自噬 (Autophagy) 是細胞在面對生存壓力時所進行的代謝過程,透過重新排列內膜系統形成自噬體,並分解不必要的蛋白質,藉此維持生合成及代謝等正常功能。但目前有研究顯示,不論在細胞試驗還是活體研究中,皆發現腸病毒71型 (EV71) 會透過誘發細胞自噬來幫助病毒的複製。
然而關於腸病毒71型是透過何種病毒蛋白來引發細胞自噬,並經由何種機制來誘發自噬的過程尚未了解。我們探討了幾個已知會影響內膜系統構造和功能的病毒蛋白2BC、2B、2C和3A,藉由觀察LC3-II的表現量做為是否發生細胞自噬的指標。實驗發現,和2B、2C以及3A組相比,2BC病毒蛋白能刺激細胞產生最多的LC3-II。此結果也在EGFP-LC3 puncta生成實驗,得到進一步的確認,表現2BC蛋白會產生數量最多的puncta。
先前在本實驗室的研究指出,腸病毒71型會藉由活化CaMKK-β並磷酸化下游AMPK,進而啟動細胞自噬。所以接下來進一步探討了病毒蛋白2BC使細胞產生自噬體的機制,我們利用Fluo-8細胞內鈣離子探針觀察細胞內鈣離子濃度變化,發現表現病毒蛋白2BC會提升細胞內鈣離子濃度。而加入了細胞內鈣離子螯合劑BAPTA-AM,CaMKK-β抑制劑STO-609,以及AMPK抑制劑Compound C後,皆大幅減少了EGFP-LC3 puncta的生成。因此認為2BC蛋白會透過CaMKK-β-AMPK這條路徑使細胞產生自噬體。另外,本篇研究也探討了腸病毒71型2BC在細胞內的表現位置,得知2BC蛋白會表現在高基氏體上,此發現也為病毒蛋白2BC可能會使高基氏體內鈣離子流入細胞質提供了進一步的證據。
總結來說,上述實驗結果支持了腸病毒71型感染後,會藉由表現2BC蛋白在高基氏體上,並造成細胞質內鈣離子濃度上升,進而活化CaMKK-β-AMPK路徑使宿主細胞產生自噬體。

Autophagy is a catabolic process involving an intracellular membrane rearrangement that recycles cellular components for cellular homeostasis. EV71 has been reported to subvert autophagy to enhance viral replication both in vitro and in vivo; however, the upstream triggers that initiate the autophagy process have not been elucidated. In this study, we attempted to identify the viral triggers that initiate autophagy, and the signaling pathway that mediates the process.
We investigated the viral proteins reported to mediate membrane remodeling in enterovirus-infected cells. Viral 2BC expression induced the highest level of microtubule-associated protein light chain 3 (LC3)-2, a hallmark of autophagosome formation, compared with those induced by viral 2B, 2C and 3A. The result was confirmed by higher number of EGFP-LC3 puncta formed in the 2BC-transfected cells. Our previous study showed that EV71 may induce autophagy through calcium/calmodulin-dependent kinase kinase-β (CaMKK-β)/adenosine monophosphate-activated protein kinase (AMPK) signaling. Therefore, to study mechanism of autophagosome formation triggered by viral 2BC expression, we measured the cytosolic calcium influx by Fluo-8 staining and found that 2BC expression raised the cytosolic calcium concentration. EGFP-LC3 punta formation in the viral 2BC expressing-cells were significantly decreased by calcium chelator, CaMKK-β inhibitor and AMPK inhibitor, supporting that 2BC triggered autophagy flux may mediated by CaMKK-β-AMPK pathway. In addition, we showed that viral 2BC was localized at Golgi apparatus, supporting the role that viral 2BC plays in mediating calcium flow from Golgi apparatus to the cytoplasm. In sum, our data supported that EV71 infection initiates autophagy through viral 2BC protein that localize to Golgi apparatus and elevates cytosolic Ca2+, a scenario that activates the CaMKK-β-AMPK pathway and consequent autophagosome formation.

目錄 1
中文摘要 3
Abstract 4
第一章 緒論 6
第一節 腸病毒的歷史 6
第二節 腸病毒的特性 7
第三節 腸病毒71型 11
第四節 正股RNA病毒與細胞自噬 13
第五節 鈣離子與細胞自噬 17
第六節 研究動機與方向 19

第二章 材料與方法 20
第一節 實驗材料 20
第二節 實驗方法 28
第三章 實驗結果 40
第一節 腸病毒71型病毒蛋白2BC促進細胞自噬 40
第二節 腸病毒71型病毒蛋白造成細胞內鈣離子的流動 42
第三節 腸病毒71型蛋白2BC啟動細胞自噬之路徑 43
第四節 腸病毒71型蛋白2BC及3A之表現位置 44
第四章 討論 45
第五章 圖表 47
第六章 參考文獻 55
第七章 附錄 61
附錄一、小RNA病毒科分類 61
附錄二、腸病毒的結構 62
附錄三、腸病毒基因體組成 62
附錄四、腸病毒的複製週期 63
附錄五、細胞自噬過程 63
附錄六、研究細胞自噬的方法 64
附錄七、鈣離子相關之細胞自噬路徑 65
附錄八、細胞內鈣離子通道 65
附錄九、病毒蛋白質調控細胞鈣離子 66
附錄十、pflag-CMV2載體 67
附錄十一、pEGFP-LC3 質體 67
附錄十二、pEFDEST51-EV71-2B質體 68

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