神經壞死病毒 (Nervous necrosis virus)，會感染重要經濟魚種並導致其死亡，如石斑魚等，造成水產養殖業的重大經濟損失。病毒本身之蛋白α 雖能誘發細胞凋亡，但其詳細機制並未清楚，因此本論文將深入探討神經壞死病毒蛋白α 所誘發石斑魚細胞凋亡之分子機制。
首先利用Annexin-Ⅴ 及TUNEL assay 來證實Red-spotted grouper nervous necrosis virus (RGNNV) 蛋白α 所誘導之早期及中期之細胞凋亡特徵，接著利用Propidium iodide (PI) 和Acridine orange (AO) / Ethidium Bromide (EtBr) 染色來證實蛋白α 所誘導之後凋亡細胞壞死現象。進一步利用JC-1 染劑可染出粒線體膜電位失衡現象和西方墨點法證實cytochrome c 之釋出，因此進一步證實蛋白α 與粒線體功能之消失有關，同時蛋白α也能活化石斑魚細胞株內之caspase-8 與caspase-3。
此外，利用基因knockdown 方式之小片段干擾性RNA (siRNA) 可有效阻斷七成蛋白α 之表現，且可減少三成病毒造成的細胞壞死及減緩病毒複製。
因此於本論文中，證實蛋白α 能誘發細胞凋亡與後凋亡細胞壞死現象。在凋亡機制方面，我們發現蛋白α 誘導之細胞粒線體膜電位失衡與cytochrome c 之釋放有關，進而導致caspase-8 與caspase-3 之活化。最後發現RNA2 之siRNA 的使用能減緩病毒導致之細胞壞死與病毒複製。
本論文針對蛋白α 凋亡機制之研究，希望能對於明瞭神經壞死病毒致病機制和疾病控制有所幫助。

Red-spotted grouper nervous necrosis virus (RGNNV) belongs to a member of the betanodaviruses that infects economically important fishes such as grouper. In the previous study, NNV protein α could induce caspases-8 and -3 activation in RNA2-transfected apoptotic cells. However, the molecular death mechanism of RGNNV protein α induced grouper cell death is still unclear.
In the present, protein α-induced grouper fin (GF-1) early and mid-apoptotic cell death could be identified by Annexin-V and TUNEL assay. Then, at the late apoptotic stage, cells enter post-apoptotic necrosis, as detected by the Propidium iodide (PI) and Acridine orange (AO) / Ethidium Bromide (EtBr) staining. Moreover, we found that protein α may induce the loss of mitochondria membrane potential and release of cytochrome c, which is correlated to activate the caspase-8 and caspase-3.
Furthermore, siRNA approach is used to knock down RNA2 expression in transient and GF-1 stable expression cell line, which can enhance the cell viability during NNV infection.
Taken our results suggest that RGNNV protein α induces post-apoptotic necrosis death via mitochondria-mediated pathway, which is required for cytochrome c release on activation of caspases-8 and -3 in fish cells. This finding may thus provide an important insight into the understanding of RGNNV pathogenesis and disease control.

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